The "amyloid cascade hypothesis" for Alzheimer's disease (AD) pathogenesis, highlights the accumulation of amyloid-β (Aβ) as a crucial trigger for the pathology. However, AD is an extremely complex disease influenced by multiple pathophysiological processes, making it impossible to attribute its onset to a single hypothesis. The endocytic pathway, where the amyloidogenic processing of APP occurs, has emerged as a pathogenic "hub" for AD. In this study, we found altered homeostasis and dynamics of endolysosomal compartments in fibroblasts from patients affected by a genetic form of AD (APP V717I mutation). These alterations corresponded to an abnormal trafficking of APP along the endolysosomal pathway, favouring its amyloidogenic processing. The identification of APP interactors involved in its trafficking and processing, and finding molecules able to interfere with these interactions, represents a promising therapeutic approach. However, the role of endosomal pathway and the possibility of modulating APP processing through it remains elusive. Among the proteins participating to APP metabolism, the RPSA receptor and its inhibitor molecule NSC47924 have been identified. In this study, we found that the inhibitor, likely by displacing APP from interaction with its receptor, reduced APP accumulation in EEs in AD cells, finally restoring both endosomal dynamics and APP distribution to those of unaffected cells. We also demonstrated that RPSA inhibition affected the aberrant APP cleavage, as it reduced the production of both APP-βCTF (C-Terminal Fragment) and Aβ in AD fibroblasts. These results highlight significant differences in endolysosomal compartments and APP processing in AD-affected cells, refining our understanding of APP/RPSA intersection.

Focusing on the early stages of Alzheimer's disease (AD) holds great promise. However, the specific events in neural cells preceding AD onset remain elusive. To address this, we utilized human-induced pluripotent stem cells carrying APPswe mutation to explore the initial changes associated with AD progression. We observed enhanced neural activity and early neuronal differentiation in APPswe cerebral organoids cultured for one month. This phenomenon was also evident when neural progenitor cells (NPCs) were differentiated into neurons. Furthermore, transcriptomic analyses of NPCs and neurons confirmed altered expression of neurogenesis-related genes in APPswe NPCs. We also found that the upregulation of reactive oxygen species (ROS) is crucial for early neuronal differentiation in these cells. In addition, APPswe neurons remained immature after initial differentiation with increased susceptibility to toxicity, providing valuable insights into the premature exit from the neural progenitor state and the increased vulnerability of neural cells in AD.

Alzheimer's Disease (AD) is the most common cause of dementia, afflicting 55 million individuals worldwide, with limited treatment available. Current AD models mainly focus on familial AD (fAD), which is due to genetic mutations. However, models for studying sporadic AD (sAD), which represents over 95% of AD cases without specific genetic mutations, are severely limited. Moreover, the fundamental species differences between humans and animals might significantly contribute to clinical failures for AD therapeutics that have shown success in animal models, highlighting the urgency to develop more translational human models for studying AD, particularly sAD. In this study, we developed a complex human pluripotent stem cell (hPSC)-based vascularized neuroimmune organoid model, which contains multiple cell types affected in human AD brains, including human neurons, microglia, astrocytes, and blood vessels. Importantly, we demonstrated that brain extracts from individuals with sAD can effectively induce multiple AD pathologies in organoids four weeks post-exposure, including amyloid beta (Aβ) plaque-like aggregates, tau tangle-like aggregates, neuroinflammation, elevated microglial synaptic pruning, synapse/neuronal loss, and impaired neural network activity. Proteomics analysis also revealed disrupted AD-related pathways in our vascularized AD neuroimmune organoids. Furthermore, after treatment with Lecanemab, an FDA-approved antibody drug targeting Aβ, AD brain extracts exposed organoids showed a significant reduction of amyloid burden, along with an elevated vascular inflammation response. Thus, the vascularized neuroimmune organoid model provides a unique opportunity to study AD, particularly sAD, under a pathophysiological relevant three-dimensional (3D) human cell environment. It also holds great promise to facilitate AD drug development, particularly for immunotherapies.

Brain organoids are an emerging in vitro 3D brain model that is integrated from pluripotent stem cells. This model mimics the human brain's developmental process and disease-related phenotypes to a certain extent while advancing the development of human brain-based biological intelligence. However, many limitations of brain organoid culture (e.g., lacking a functional vascular system, etc.) prevent in vitro-cultured organoids from truly replicating the human brain in terms of cell type and structure. To improve brain organoids' scalability, efficiency, and stability, this paper discusses important contributions of material biology and microprocessing technology in solving the related limitations of brain organoids and applying the latest imaging technology to make real-time imaging of brain organoids possible. In addition, the related applications of brain organoids, especially the development of organoid intelligence combined with artificial intelligence, are analyzed, which will help accelerate the rational design and guidance of brain organoids.

Progressive Supranuclear Palsy (PSP) is a severe neurodegenerative disease characterized by tangles of hyperphosphorylated tau protein and tufted astrocytes. Developing treatments for PSP is challenging due to the lack of disease models reproducing its key pathological features. This study aimed to model sporadic PSP-Richardson's syndrome (PSP-RS) using multi-donor midbrain organoids (MOs).

The MOs were generated by pooling induced pluripotent stem cells (iPSCs) from 4 patients with sporadic probable PSP-RS and compared them with MOs from 3 healthy control (HC) subjects. We performed comprehensive analyses of MOs over 120 days to assess neuronal death, reactive gliosis, and the accumulation of 4R-tau and hyperphosphorylated tau forms (pThr231, pSer396, pThr181, and pSer202/pThr205 [AT8]) using immunofluorescence microscopy and Western blot. On day 90, immunohistochemical analysis using pSer396 and AT8 antibodies was conducted to assess disease pathology.

PSP-derived MOs showed progressive size reduction compared with HC-derived MOs, linked to upregulated apoptosis-related mRNA markers. Dopaminergic neuron degeneration was marked by decreased tyrosine hydroxylase (TH) and increased neurofilament light chain (NfL). Immunofluorescence and Western blot revealed accumulation of all investigated tau forms with a peak at 90 days, along with a significant rise in GFAP-positive cells in PSP-derived MOs. Immunochemistry confirmed typical PSP histological alterations, such as neurofibrillary tangles and tufted-shaped astrocytes, absent in HC-derived organoids.

We developed a robust in vitro PSP model reproducing the key molecular and histologic features of the disease. This result holds promise for advancing basic and clinical research in PSP, paving the way for in vitro molecular diagnosis and identification of novel therapeutic targets. ANN NEUROL 2025;97:845-859.

Over the past two decades, significant advancements have been made in the induced pluripotent stem cell (iPSC) technology. These developments have enabled the broader application of iPSCs in neuroscience, improved our understanding of disease pathogenesis, and advanced the investigation of therapeutic targets and methods. Specifically, optimizations in reprogramming protocols, coupled with improved neuronal differentiation and maturation techniques, have greatly facilitated the generation of iPSC-derived neural cells. The integration of the cerebral organoid technology and CRISPR/Cas9 genome editing has further propelled the application of iPSCs in neurodegenerative diseases to a new stage. Patient-derived or CRISPR-edited cerebral neurons and organoids now serve as ideal disease models, contributing to our understanding of disease pathophysiology and identifying novel therapeutic targets and candidates. In this review, we examine the development of iPSC-based models in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.

Long-term preservation of fully differentiated human neurons poses a longstanding challenge in neuroscience research. Numerous cellular disease models have been established using cultured human neuronal cells, including our three-dimensional (3D) human neural cell culture model of Alzheimer's disease (AD). However, the absence of a reliable method for preserving fully differentiated human neural cell cultures for a long time has hindered the sharing and standardization of these models. To address this critical limitation, we focused on cryopreservation, which is the gold standard for long-term preservation, and combined this with three key technological advancements. First, we employed parallelized microfluidic devices for the efficient generation of 3D cell cultures within uniform hydrogel microbeads (~ 220 μm), which facilitate the rapid exchange of media ingredients and cryoprotectants. Second, we implemented a cytophobic microwell system to safeguard neuron-encapsulated microbeads from fusion and aggregation. Third, we developed a novel inducible AD cell model optimized for cryopreservation and AD drug testing. We have successfully maintained encapsulated control and AD neural progenitor cells in microwells during differentiation for 12 days. Notably, fully differentiated human neural cells can be cryopreserved within Matrigel microbeads while retaining intact and mature neuronal processes, exhibiting no signs of damage to neurites following freeze/thaw cycles. Furthermore, we have demonstrated the successful cryopreservation, thawing, and induction of pathogenic Amyloid-β 42 (Aβ42) generation in fully differentiated AD neural progenitor cells. Our study offers a solution for one of the major challenges in neuroscience research, utilizing porous hydrogel microbead structures to facilitate rapid delivery of cryoprotectants and protect complex neuronal structures without undergoing damaging cell dissociation steps. The inducible "3D human microbead model of AD" enhances the speed, efficacy, and reproducibility of AD drug screening.

Neuroinflammation is a complex immune response triggered by brain injury or pathological stimuli, and is highly exacerbated in neurodegenerative diseases. It plays a dual role in the central nervous system, promoting repair in acute stages while aggravating disease progression by contributing to neuronal loss, synaptic dysfunction, and glial dysregulation in chronic phases. Inflammatory responses are mainly orchestrated by microglia and infiltrated monocytes, which, when dysregulated, not only harm existing neurons, but also impair the survival and differentiation of neural stem and progenitor cells in the affected brain regions. Modulating neuroinflammation is crucial for harnessing its protective functions while minimizing its detrimental effects. Current therapeutic strategies focus on fine-tuning inflammatory responses through pharmacological agents, bioactive molecules, and stem cell-based therapies. These approaches aim to restore immune homeostasis, support neuroprotection, and promote regeneration in various neurological disorders. However, animal models sometimes fail to reproduce human-specific inflammatory responses in the brain. In this context, stem-cell-derived models provide a powerful tool to study neuroinflammatory mechanisms in a patient-specific and physiologically relevant context. These models facilitate high-throughput screening, personalized medicine, and the development of targeted therapies while addressing the limitations of traditional animal models, paving the way for more targeted and effective treatments.

The design of neural tissue models with architectural and biochemical relevance to native tissues opens the way for the fundamental study and development of therapies for many disorders with limited treatment options. Here, we systematically review the most recent literature on 3D bioprinted neural models, including their potential for use in drug screening. Neural tissues that model the central nervous system (CNS) from the relevant literature are reviewed with comprehensive summaries of each study, and discussion of the model types, bioinks and additives, cell types used, bioprinted construct shapes and culture time, and the characterization methods used. In this review, we accentuate the lack of standardization among characterization methods to analyze the functionality (including chemical, metabolic and other pathways) and mechanical relevance of the 3D bioprinted constructs, and discuss this as a critical area for future exploration. These gaps must be addressed for this technology to be applied for effective drug screening applications, despite its enormous potential for rapid and efficient drug screening. The future of biomimetic, 3D printed neural tissues is promising and evaluation of the in vivo relevance on multiple levels should be sought to adequately compare model performance and develop viable treatment options for neurodegenerative diseases, or other conditions that affect the CNS.

Human brain organoids produce anatomically relevant cellular structures and recapitulate key aspects of in vivo brain function, which holds great potential to model neurological diseases and screen therapeutics. However, the long growth time of 3D systems complicates the culturing of brain organoids and results in heterogeneity across samples hampering their applications. We developed an integrated platform to enable robust and long-term culturing of 3D brain organoids. We designed a mesofluidic bioreactor device based on a reaction-diffusion scaling theory, which achieves robust media exchange for sufficient nutrient delivery in long-term culture. We integrated this device with longitudinal tracking and machine learning-based classification tools to enable non-invasive quality control of live organoids. This integrated platform allows for sample pre-selection for downstream molecular analysis. Transcriptome analyses of organoids revealed that our mesofluidic bioreactor promoted organoid development while reducing cell death. Our platform thus offers a generalizable tool to establish reproducible culture standards for 3D cellular systems for a variety of applications beyond brain organoids.

Human cardiac organoids closely replicate the architecture and function of the human heart, offering a potential accurate platform for studying cellular and molecular features of aging cardiomyopathy. Senolytics have shown potential in addressing age-related pathologies but their potential to reverse aging-related human cardiomyopathy remains largely unexplored.

We employed human iPSC-derived cardiac organoids (hCOs/hCardioids) to model doxorubicin(DOXO)-induced cardiomyopathy in an aged context. hCardioids were treated with DOXO and subsequently with a combination of two senolytics: dasatinib (D) and quercetin (Q).

DOXO-treated hCardioids exhibited significantly increased oxidative stress, DNA damage (pH2AX), cellular senescence (p16INK4A) and decreased cell proliferation associated with a senescence-associated secretory phenotype (SASP). DOXO-treated hCardioids were considerably deprived of cardiac progenitors and displayed reduced cardiomyocyte proliferation as well as contractility. These distinctive aging-associated characteristics were confirmed by global RNA-sequencing analysis. Treatment with D+Q reversed these effects, reducing oxidative stress and senescence markers, alleviating SASP, and restoring hCardioids viability and function. Additionally, senolytics replenished cardiac progenitors and reversed the cardiomyocyte proliferation deficit.

Doxorubicin triggers an age-associated phenotype in hCardioids reliably modelling the main cellular and molecular features of aging cardiomyopathy. Senescence is a key mechanism of the aged-hCOs phenotype as senolytics rejuvenated aged-hCardioids restoring their structure and function while reverting the age-associated regenerative deficit.

Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.

Peripheral nerve injury (PNI) resulting in lesions is highly prevalent clinically, but current therapeutic approaches fail to provide satisfactory outcomes in many patients. While peripheral nerves have intrinsic regenerative capacity, the regenerative capabilities of peripheral nerves are often insufficient to restore full functionality. This highlights an unmet need for developing more effective strategies to repair damaged peripheral nerves and improve regenerative success. Consequently, researchers are actively exploring a variety of therapeutic strategies, encompassing the local delivery of trophic factors or bioactive molecules, the design of advanced biomaterials that interact with regenerating axons, and augmentation with nerve guidance conduits or complex prostheses. However, clinical translation of these technologies remains limited, emphasizing the need for continued research on peripheral nerve regeneration modalities that can enhance functional restoration. Experimental models that accurately recapitulate key aspects of peripheral nerve injury and repair biology can accelerate therapeutic development by enabling systematic testing of new techniques. Advancing regenerative therapies for PNI requires bridging the gap between basic science discoveries and clinical application. This review discusses different in vitro models of peripheral nerve injury and repair, including their advantages, limitations, and potential applications.

The tauopathies are defined by pathological tau protein aggregates within a spectrum of clinically heterogeneous neurodegenerative diseases. The primary tauopathies meet the definition of rare diseases in the United States. There is no approved treatment for primary tauopathies. In this context, designing the most efficient development programs to translate promising targets and treatments from preclinical studies to early-phase clinical trials is vital. In September 2022, the Rainwater Charitable Foundation convened an international expert workshop focused on the translation of tauopathy therapeutics through early-phase trials. Our report on the workshop recommends a framework for principled drug development and a companion lexicon to facilitate communication focusing on reproducibility and achieving common elements. Topics include the selection of targets, drugs, biomarkers, participants, and study designs. The maturation of pharmacodynamic biomarkers to demonstrate target engagement and surrogate disease biomarkers is a crucial unmet need. HIGHLIGHTS: Experts provided a framework to translate therapeutics (discovery to clinical trials). Experts focused on the "5 Rights" (target, drug, biomarker, participants, trial). Current research on frontotemporal degeneration, progressive supranuclear palsy, and corticobasal syndrome therapeutics includes 32 trials (37% on biologics) Tau therapeutics are being tested in Alzheimer's disease; primary tauopathies have a large unmet need.

Advanced brain organoids provide promising platforms for deciphering the cellular and molecular processes of human neural development and diseases. Although various studies and reviews have described developments and advancements in brain organoids, few studies have comprehensively summarized and analyzed the global trends in this area of neuroscience. To identify and further facilitate the development of cerebral organoids, we utilized bibliometrics and visualization methods to analyze the global trends and evolution of brain organoids in the last 10 years. First, annual publications, countries/regions, organizations, journals, authors, co-citations, and keywords relating to brain organoids were identified. The hotspots in this field were also systematically identified. Subsequently, current applications for brain organoids in neuroscience, including human neural development, neural disorders, infectious diseases, regenerative medicine, drug discovery, and toxicity assessment studies, are comprehensively discussed. Towards that end, several considerations regarding the current challenges in brain organoid research and future strategies to advance neuroscience will be presented to further promote their application in neurological research.

Despite the potential toxicity of commercial chemicals to the development of the nervous system (known as developmental neurotoxicity or DNT), conventionalin vitrocell models have primarily been employed for the assessment of acute neuronal toxicity. On the other hand, animal models used for the assessment of DNT are not physiologically relevant due to the heterogenic difference between humans and animals. In addition, animal models are low-throughput, time-consuming, expensive, and ethically questionable. Recently, human brain organoids have emerged as a promising alternative to assess the detrimental effects of chemicals on the developing brain. However, conventional organoid culture systems have several technical limitations including low throughput, lack of reproducibility, insufficient maturity of organoids, and the formation of the necrotic core due to limited diffusion of nutrients and oxygen. To address these issues and establish predictive DNT models, cerebral organoids were differentiated in a dynamic condition in a unique pillar/perfusion plate, which were exposed to test compounds to evaluate DNT potential. The pillar/perfusion plate facilitated uniform, dynamic culture of cerebral organoids with improved proliferation and maturity by rapid, bidirectional flow generated on a digital rocker. Day 9 cerebral organoids in the pillar/perfusion plate were exposed to ascorbic acid (DNT negative) and methylmercury (DNT positive) in a dynamic condition for 1 and 3 weeks, and changes in organoid morphology and neural gene expression were measured to determine DNT potential. As expected, ascorbic acid did not induce any changes in organoid morphology and neural gene expression. However, exposure of day 9 cerebral organoids to methylmercury resulted in significant changes in organoid morphology and neural gene expression. Interestingly, methylmercury did not induce adverse changes in cerebral organoids in a static condition, thus highlighting the importance of dynamic organoid culture in DNT assessment.

The maintenance and expansion of human neural stem cells (hNSCs) in 3D tissue scaffolds is a promising strategy in producing cost-effective hNSCs with quality and quantity applicable for clinical applications. A few biopolymers have been extensively used to fabricate 3D scaffolds, including hyaluronic acid, collagen, alginate, and chitosan, due to their bioactive nature and availability. However, these polymers are usually applied in combination with other biomolecules, leading to their responses difficult to ascribe to. Here, scaffolds made of chitosan, alginate, hyaluronic acid, or collagen, are explored for hNSC expansion under xeno-free and chemically defined conditions and compared for hNSC multipotency maintenance. This study shows that the scaffolds made of pure chitosan support the highest adhesion and growth of hNSCs, yielding the most viable cells with NSC marker protein expression. In contrast, the presence of alginate, hyaluronic acid, or collagen induces differentiation toward immature neurons and astrocytes even in the maintenance medium and absence of differentiation factors. The cells in pure chitosan scaffolds preserve the level of transmembrane protein profile similar to that of standard culture. These findings point to the potential of using pure chitosan scaffolds as a base scaffolding material for hNSC expansion in 3D.

Brain organoids provide a unique opportunity to model organ development in a system similar to human organogenesis in vivo. Brain organoids thus hold great promise for drug screening and disease modeling. Conventional approaches to organoid characterization predominantly rely on molecular analysis methods, which are expensive, time-consuming, labor-intensive, and involve the destruction of the valuable three-dimensional (3D) architecture of the organoids. This reliance on end-point assays makes it challenging to assess cellular and subcellular events occurring during organoid development in their 3D context. As a result, the long developmental processes are not monitored nor assessed. The ability to perform non-invasive assays is critical for longitudinally assessing features of organoid development during culture. In this paper, we demonstrate a label-free high-content imaging approach for observing changes in organoid morphology and structural changes occurring at the cellular and subcellular level. Enabled by microfluidic-based culture of 3D cell systems and a novel 3D quantitative phase imaging method, we demonstrate the ability to perform non-destructive high-resolution quantitative image analysis of the organoid. The highlighted results demonstrated in this paper provide a new approach to performing live, non-destructive monitoring of organoid systems during culture.

Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized pathologically by extracellular deposition of β-amyloid (Aβ) into senile plaques and intracellular accumulation of hyperphosphorylated tau (pTau) as neurofibrillary tangles. Clinically, AD patients show memory deterioration with varying cognitive dysfunctions. The exact molecular mechanisms underlying AD are still not fully understood, and there are no efficient drugs to stop or reverse the disease progression. In this review, we first provide an update on how the risk factors, including APOE variants, infections and inflammation, contribute to AD; how Aβ and tau become abnormally accumulated and how this accumulation plays a role in AD neurodegeneration. Then we summarize the commonly used experimental models, diagnostic and prediction strategies, and advances in periphery biomarkers from high-risk populations for AD. Finally, we introduce current status of development of disease-modifying drugs, including the newly officially approved Aβ vaccines, as well as novel and promising strategies to target the abnormal pTau. Together, this paper was aimed to update AD research progress from fundamental mechanisms to the clinical diagnosis and therapies.

Reduction of adult hippocampal neurogenesis is an early critical event in Alzheimer's disease (AD), contributing to progressive memory loss and cognitive decline. Reduced levels of the nucleoporin 153 (Nup153), a key epigenetic regulator of NSC stemness, characterize the neural stem cells isolated from a mouse model of AD (3×Tg) (AD-NSCs) and determine their altered plasticity and gene expression.

Nup153-regulated mechanisms contributing to NSC function were investigated: (1) in cultured NSCs isolated from AD and wild type (WT) mice by proteomics; (2) in vivo by lentiviral-mediated delivery of Nup153 or GFP in the hippocampus of AD and control mice analyzing neurogenesis and cognitive function; (3) in human iPSC-derived brain organoids obtained from AD patients and control subjects as a model of neurodevelopment.

Proteomic approach identified Nup153 interactors in WT- and AD-NSCs potentially implicated in neurogenesis regulation. Gene ontology (GO) analysis showed that Nup153-bound proteins in WT-NSCs were involved in RNA metabolism, nuclear import and epigenetic mechanisms. Nup153-bound proteins in AD-NSCs were involved in pathways of neurodegeneration, mitochondrial dysfunction, proteasomal processing and RNA degradation. Furthermore, recovery of Nup153 levels in AD-NSCs reduced the levels of oxidative stress markers and recovered proteasomal activity. Lentiviral-mediated delivery of Nup153 in the hippocampal niche of AD mice increased the proliferation of early progenitors, marked by BrdU/DCX and BrdU/PSANCAM positivity and, later, the integration of differentiating neurons in the cell granule layer (BrdU/NeuN+ cells) compared with GFP-injected AD mice. Consistently, Nup153-injected AD mice showed an improvement of cognitive performance in comparison to AD-GFP mice at 1 month after virus delivery assessed by Morris Water Maze. To validate the role of Nup153 in neurogenesis we took advantage of brain organoids derived from AD-iPSCs characterized by fewer neuroepithelial progenitor loops and reduced differentiation areas. The upregulation of Nup153 in AD organoids recovered the formation of neural-like tubes and differentiation.

Our data suggest that the positive effect of Nup153 on neurogenesis is based on a complex regulatory network orchestrated by Nup153 and that this protein is a valuable disease target.

Microglia, the resident immune-competent cells of the brain, become dysfunctional in Alzheimer's disease (AD), and their aberrant immune responses contribute to the accumulation of pathological proteins and neuronal injury. Genetic studies implicate microglia in the development of AD, prompting interest in developing immunomodulatory therapies to prevent or ameliorate disease. However, microglia take on diverse functional states in disease, playing both protective and detrimental roles in AD, which largely overlap and may shift over the disease course, complicating the identification of effective therapeutic targets. Extensive evidence gathered using transgenic mouse models supports an active role of microglia in pathology progression, though results vary and can be contradictory between different types of models and the degree of pathology at the time of study. Here, we review microglial immune signaling and responses that contribute to the accumulation and spread of pathological proteins or directly affect neuronal health. We additionally explore the use of induced pluripotent stem cell (iPSC)-derived models to study living human microglia and how they have contributed to our knowledge of AD and may begin to fill in the gaps left by mouse models. Ultimately, mouse and iPSC-derived models have their own limitations, and a comprehensive understanding of microglial dysfunction in AD will only be established by an integrated view across models and an appreciation for their complementary viewpoints and limitations.

Research in the field of traumatic brain injury has until now heavily relied on the use of animal models to identify potential therapeutic approaches. However, a long series of failed clinical trials has brought many scientists to question the translational reliability of pre-clinical results obtained in animals. The search for an alternative to conventional models that better replicate human pathology in traumatic brain injury is thus of the utmost importance for the field. Recently, orthotopic xenotransplantation of human brain organoids into living animal models has been achieved. This review summarizes the existing literature on this new method, focusing on its potential applications in preclinical research, both in the context of cell replacement therapy and disease modelling. Given the obvious advantages of this approach to study human pathologies in an in vivo context, we here critically review its current limitations while considering its possible applications in traumatic brain injury research.

Alzheimer disease (AD) is a heterogenous and multifactorial disease, and its pathology is partly driven by microglia and their activated phenotype. Brain organoids (BOs) are gaining prominence as a relevant model of the human brain for the study of AD; however, BOs are commonly devoid of microglia. To overcome this limitation, current protocols incorporate microglia through either (1) co-culture (BO co-culture), or (2) molecular manipulation at critical windows of BO development to have microglia arise innately (BO innate cultures). It is currently unclear whether the microglia incorporated into BOs by either of these two protocols differ in function.

At in vitro day 90, BO innate cultures and BO-co-cultures were challenged with the AD-related β-amyloid peptide (Aβ) for up to 72 h. After Aβ challenge, BOs were collected for immunoblotting. Immunoblots compared immunodensity and protein banding of Aβ and ionized calcium-binding adapter molecule 1 (IBA1, a marker of microglial activation) in BOs. The translational potential of these observations was supported using 56 human cortical samples from neurocognitively normal donors and patients with early-onset AD and late-onset AD. Statistical analyses were conducted using the Kruskal-Wallis test, a two-way ANOVA, or a simple linear regression, and where applicable, followed by Dunn's or Sidak's test.

We show that BO co-cultures promote Aβ oligomerization as early as 24 h and this coincides with a significant increase in IBA1 levels. In contrast, the Aβs do not oligomerize in BO innate cultures and the IBA1 response was modest and only emerged after 48 h. In human cortical samples, we found IBA1 levels correlated with age at onset, age at death, and the putative diagnostic Aβ(1-42)/Aβ(1-40) ratio (particularly in their oligomeric forms) in a sex-dependent manner.

Our unique observations suggest that BOs with innate microglia model the response of a healthy brain to Aβ, and by extension the initial stages of Aβ challenge. It would be impossible to model these early stages of pathogenesis in BOs where microglia are already compromised, such as those with microglia incorporated by co-culture.

Damage to the central nervous system (CNS) often leads to irreversible neurological deficits, and there are currently few effective treatments available. However, recent advancements in regenerative medicine have identified CNS organoids as promising therapeutic options for addressing CNS injuries. These organoids, composed of various neurons and supporting cells, have shown potential for direct repair at injury sites. CNS organoids resemble the structure and function of actual brain tissue, which allows them to adapt and function well within the physiological environment when transplanted into injury sites. Research findings suggest that CNS organoids can replace damaged neurons, form new neural connections, and promote neural recovery. This review highlights the emerging benefits, evaluates preclinical transplantation outcomes, and explores future strategies for optimizing neuroregeneration using CNS organoids. With continued research and technological advancements, these organoids could provide new hope for patients suffering from neurological deficits.

Recent advances in human pluripotent stem cell (hPSC) technologies have prompted the emergence of new research fields and applications for human neurons and brain organoids. Brain organoids have gained attention as an in vitro model system that recapitulates the higher structure, cellular diversity and function of the brain to explore brain development, disease modeling, drug screening, and regenerative medicine. This progress has been accelerated by abundant interactions of brain organoid technology with various research fields. A cross-disciplinary approach with human brain organoid technology offers a higher-ordered advance for more accurately understanding the human brain. In this review, we summarize the status of neural induction in two- and three-dimensional culture systems from hPSCs and the modeling of neurodegenerative diseases using brain organoids. We also highlight the latest bioengineered technologies for the assembly of spatially higher-ordered neural tissues and prospects of brain organoid technology toward the understanding of the potential and abilities of the human brain.

Late-onset Alzheimer's disease (LOAD) is the most common form of Alzheimer's disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-β (Aβ) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)-based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aβ-dependent neurodegeneration, and treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aβ deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

Age-related neurodegenerative diseases, like Alzheimer's disease (AD), are challenging diseases for those affected with no cure and limited treatment options. Functional, human derived brain tissues that represent the diverse genetic background and cellular subtypes contributing to sporadic AD (sAD) are limited. Human stem cell derived brain organoids recapitulate some features of human brain cytoarchitecture and AD-like pathology, providing a tool for illuminating the relationship between AD pathology and neural cell dysregulation leading to cognitive decline. In this review, we explore current strategies for implementing brain organoids in the study of AD as well as the challenges associated with investigating age-related brain diseases using organoid models.

Rare genetic diseases with neurodevelopmental disorders (NDDs) encompass several heterogeneous conditions (autism spectrum disorder (ASD), intellectual disability (ID), attention deficit hyperactivity disorder (ADHD), specific learning disorder (SLD), among others). Currently, few treatments are available for these patients. The difficulty in accessing human brain samples and the discrepancies between human and animal models highlight the need for new research approaches. One promising approach is the use of the cerebral organoids. These 3D, self-organized structures, generated from induced pluripotent stem cells (iPSCs), enable the reproduction of the stages of human brain development, from the proliferation of neural stem cells to their differentiation into neurons, oligodentrocytes, and astrocytes. Cerebral organoids hold great promise in understanding brain development and in the search for treatments.

Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.

Human brain organoids produce anatomically relevant cellular structures and recapitulate key aspects of in vivo brain function, which holds great potential to model neurological diseases and screen therapeutics. However, the long growth time of 3D systems complicates the culturing of brain organoids and results in heterogeneity across samples hampering their applications. We developed an integrated platform to enable robust and long-term culturing of 3D brain organoids. We designed a mesofluidic bioreactor device based on a reaction-diffusion scaling theory, which achieves robust media exchange for sufficient nutrient delivery in long-term culture. We integrated this device with longitudinal tracking and machine learning-based classification tools to enable non-invasive quality control of live organoids. This integrated platform allows for sample pre-selection for downstream molecular analysis. Transcriptome analyses of organoids revealed that our mesofluidic bioreactor promoted organoid development while reducing cell death. Our platform thus offers a generalizable tool to establish reproducible culture standards for 3D cellular systems for a variety of applications beyond brain organoids.

Since the emergence of the first cerebral organoid (CO) in 2013, advancements have transformed central nervous system (CNS) research. Initial efforts focused on studying the morphogenesis of COs and creating reproducible models. Numerous methodologies have been proposed, enabling the design of the brain organoid to represent specific regions and spinal cord structures. CNS organoids now facilitate the study of a wide range of CNS diseases, from infections to tumors, which were previously difficult to investigate. We summarize the major advancements in CNS organoids, concerning morphogenetic designs and disease models. We examine the development of fabrication procedures and how these advancements have enabled the generation of region-specific brain organoids and spinal cord models. We highlight the application of these organoids in studying various CNS diseases, demonstrating the versatility and potential of organoid models in advancing our understanding of complex conditions. We discuss the current challenges in the field, including issues related to reproducibility, scalability, and the accurate recapitulation of the in vivo environment. We provide an outlook on prospective studies and future directions. This review aims to provide a comprehensive overview of the state-of-the-art CNS organoid research, highlighting key developments, current challenges, and prospects in the field.

The ability to reprogram patient-derived-somatic cells to IPSCs (Induced Pluripotent Stem Cells) has led to a better understanding of aging and age-related diseases like Parkinson's, and Alzheimer's. The established patient-derived disease models mimic disease pathology and can be used to design drugs for aging and age-related diseases. However, the age and genetic mutations of the donor cells, the employed reprogramming, and the differentiation protocol might often pose challenges in establishing an appropriate disease model. In this review, we will focus on the various strategies for the successful reprogramming and differentiation of patient-derived cells to disease models for aging and age-related diseases, emphasizing the accuracy in the recapitulation of disease pathology and ways to overcome the limitations of its potential application in cell replacement therapy and drug development.

Age is the number one risk factor for developing a neurodegenerative disease (ND), such as Alzheimer's disease (AD) or Parkinson's disease (PD). With our rapidly ageing world population, there will be an increased burden of ND and need for disease-modifying treatments. Currently, however, translation of research from bench to bedside in NDs is poor. This may be due, at least in part, to the failure to account for the potential effect of ageing in preclinical modelling of NDs. While ageing can impact upon physiological response in multiple ways, only a limited number of preclinical studies of ND have incorporated ageing as a factor of interest. Here, we evaluate the aged phenotype and highlight the critical, but unmet, need to incorporate aspects of this phenotype into both the in vitro and in vivo models used in ND research. Given technological advances in the field over the past several years, we discuss how these could be harnessed to create novel models of ND that more readily incorporate aspects of the aged phenotype. This includes a recently described in vitro panel of ageing markers, which could help lead to more standardised models and improve reproducibility across studies. Importantly, we cannot assume that young cells or animals yield the same responses as seen in the context of ageing; thus, an improved understanding of the biology of ageing, and how to appropriately incorporate this into the modelling of ND, will ensure the best chance for successful translation of new therapies to the aged patient.

Neurodegenerative diseases are increasing in prevalence and comprise a large socioeconomic burden on patients and their caretakers. The need for effective therapies and avenues for disease prevention and monitoring is of paramount importance. Fluid biomarkers for neurodegenerative diseases have gained a variety of uses, including informing participant selection for clinical trials, lending confidence to clinical diagnosis and disease staging, determining prognosis, and monitoring therapeutic response. Their role is expected to grow as disease-modifying therapies start to be available to a broader range of patients and as prevention strategies become established. Many of the underlying molecular mechanisms of currently used biomarkers are incompletely understood. Animal models and in vitro systems using cell lines have been extensively employed but face important translatability limitations. Induced pluripotent stem cell (iPSC) technology, where a theoretically unlimited range of cell types can be reprogrammed from peripheral cells sampled from patients or healthy individuals, has gained prominence over the last decade. It is a promising avenue to study physiological and pathological biomarker function and response to experimental therapeutics. Such systems are amenable to high-throughput drug screening or multiomics readouts such as transcriptomics, lipidomics, and proteomics for biomarker discovery, investigation, and validation. The present review describes the current state of biomarkers in the clinical context of neurodegenerative diseases, with a focus on Alzheimer's disease and frontotemporal dementia. We include a discussion of how iPSC models have been used to investigate and test biomarkers such as amyloid-β, phosphorylated tau, neurofilament light chain or complement proteins, and even nominate novel biomarkers. We discuss the limitations of current iPSC methods, mentioning alternatives such as coculture systems and three-dimensional organoids which address some of these concerns. Finally, we propose exciting prospects for stem cell transplantation paradigms using animal models as a preclinical tool to study biomarkers in the in vivo context.

Drug discovery is a generally inefficient and capital-intensive process. For neurodegenerative diseases (NDDs), the development of novel therapeutics is particularly urgent considering the long list of late-stage drug candidate failures. Although our knowledge on the pathogenic mechanisms driving neurodegeneration is growing, additional efforts are required to achieve a better and ultimately complete understanding of the pathophysiological underpinnings of NDDs. Beyond the etiology of NDDs being heterogeneous and multifactorial, this process is further complicated by the fact that current experimental models only partially recapitulate the major phenotypes observed in humans. In such a scenario, multi-omic approaches have the potential to accelerate the identification of new or repurposed drugs against a multitude of the underlying mechanisms driving NDDs. One major advantage for the implementation of multi-omic approaches in the drug discovery process is that these overarching tools are able to disentangle disease states and model perturbations through the comprehensive characterization of distinct molecular layers (i.e., genome, transcriptome, proteome) up to a single-cell resolution. Because of recent advances increasing their affordability and scalability, the use of omics technologies to drive drug discovery is nascent, but rapidly expanding in the neuroscience field. Combined with increasingly advanced in vitro models, which particularly benefited from the introduction of human iPSCs, multi-omics are shaping a new paradigm in drug discovery for NDDs, from disease characterization to therapeutics prediction and experimental screening. In this review, we discuss examples, main advantages and open challenges in the use of multi-omic approaches for the in vitro discovery of targets and therapies against NDDs.

Alois Alzheimer described the first patient with Alzheimer's disease (AD) in 1907 and today AD is the most frequently diagnosed of dementias. AD is a multi-factorial neurodegenerative disorder with familial, life style and comorbidity influences impacting a global population of more than 47 million with a projected escalation by 2050 to exceed 130 million. In the USA the AD demographic encompasses approximately six million individuals, expected to increase to surpass 13 million by 2050, and the antecedent phase of AD, recognized as mild cognitive impairment (MCI), involves nearly 12 million individuals. The economic outlay for the management of AD and AD-related cognitive decline is estimated at approximately 355 billion USD. In addition, the intensifying prevalence of AD cases in countries with modest to intermediate income countries further enhances the urgency for more therapeutically and cost-effective treatments and for improving the quality of life for patients and their families. This narrative review evaluates the pathophysiological basis of AD with an initial focus on the therapeutic efficacy and limitations of the existing drugs that provide symptomatic relief: acetylcholinesterase inhibitors (AChEI) donepezil, galantamine, rivastigmine, and the N-methyl-D-aspartate receptor (NMDA) receptor allosteric modulator, memantine. The hypothesis that amyloid-β (Aβ) and tau are appropriate targets for drugs and have the potential to halt the progress of AD is critically analyzed with a particular focus on clinical trial data with anti-Aβ monoclonal antibodies (MABs), namely, aducanumab, lecanemab and donanemab. This review challenges the dogma that targeting Aβ will benefit the majority of subjects with AD that the anti-Aβ MABs are unlikely to be the "magic bullet". A comparison of the benefits and disadvantages of the different classes of drugs forms the basis for determining new directions for research and alternative drug targets that are undergoing pre-clinical and clinical assessments. In addition, we discuss and stress the importance of the treatment of the co-morbidities, including hypertension, diabetes, obesity and depression that are known to increase the risk of developing AD.