Therapeutic Potential of Targeting Prokineticin Receptors in Diseases.

The prokineticins (PKs) were discovered approximately 20 years ago as small peptides inducing gut contractility. Today, they are established as angiogenic, anorectic, and proinflammatory cytokines, chemokines, hormones, and neuropeptides involved in variety of physiologic and pathophysiological pathways. Their altered expression or mutations implicated in several diseases make them a potential biomarker. Their G-protein coupled receptors, PKR1 and PKR2, have divergent roles that can be therapeutic target for treatment of cardiovascular, metabolic, and neural diseases as well as pain and cancer. This article reviews and summarizes our current knowledge of PK family functions from development of heart and brain to regulation of homeostasis in health and diseases. Finally, the review summarizes the established roles of the endogenous peptides, synthetic peptides and the selective ligands of PKR1 and PKR2, and nonpeptide orthostatic and allosteric modulator of the receptors in preclinical disease models. The present review emphasizes the ambiguous aspects and gaps in our knowledge of functions of PKR ligands and elucidates future perspectives for PK research. SIGNIFICANCE STATEMENT: This review provides an in-depth view of the prokineticin family and PK receptors that can be active without their endogenous ligand and exhibits "constitutive" activity in diseases. Their non- peptide ligands display promising effects in several preclinical disease models. PKs can be the diagnostic biomarker of several diseases. A thorough understanding of the role of prokineticin family and their receptor types in health and diseases is critical to develop novel therapeutic strategies with safety concerns.

Dissecting reactive astrocyte responses: lineage tracing and morphology-based clustering.

Brain damage triggers diverse cellular and molecular events, with astrocytes playing a crucial role in activating local neuroprotective and reparative signaling within damaged neuronal circuits. Here, we investigated reactive astrocytes using a multidimensional approach to categorize their responses into different subtypes based on morphology. This approach utilized the StarTrack lineage tracer, single-cell imaging reconstruction and multivariate data analysis. Our findings identified three profiles of reactive astrocyte responses, categorized by their effects on cell size- and shape- related morphological parameters: "moderate", "strong," and "very strong". We also examined the heterogeneity of astrocyte reactivity, focusing on spatial and clonal distribution. Our research revealed a notable enrichment of protoplasmic and fibrous astrocytes within the "strong" and "very strong" response subtypes. Overall, our study contributes to a better understanding of astrocyte heterogeneity in response to an injury. By characterizing the diverse reactive responses among astrocyte subpopulations, we provide insights that could guide future research aimed at identifying novel therapeutic targets to mitigate brain damage and promote neural repair.

Leptin enhances adult neurogenesis and reduces pathological features in a transgenic mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia worldwide and is characterized by the presence of senile plaques by amyloid-beta (Aß) and neurofibrillary tangles of hyperphosphorylated Tau protein. These changes lead to progressive neuronal degeneration and dysfunction, resulting in severe brain atrophy and cognitive deficits. With the discovery that neurogenesis persists in the adult mammalian brain, including brain regions affected by AD, studies of the use of neural stem cells (NSCs) for the treatment of neurodegenerative diseases to repair or prevent neuronal cell loss have increased. Here we demonstrate that leptin administration increases the neurogenic process in the dentate gyrus of the hippocampus as well as in the subventricular zone of lateral ventricles of adult and aged mice. Chronic treatment with leptin increased NSCs proliferation with significant effects on proliferation and differentiation of newborn cells. The expression of the long form of the leptin receptor, LepRb, was detected in the neurogenic niches by reverse qPCR and immunohistochemistry. Moreover, leptin modulated astrogliosis, microglial cell number and the formation of senile plaques. Additionally, leptin led to attenuation of Aß-induced neurodegeneration and superoxide anion production as revealed by Fluoro-Jade B and dihydroethidium staining. Our study contributes to the understanding of the effects of leptin in the brain that may lead to the development of new therapies to treat Alzheimer's disease.

Neuroprotective effect of heparin Trisulfated disaccharide on ischemic stroke.

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca2+) overload. High concentrations of intracellular calcium ([Ca2+]i) can trigger cell death in the neural tissue, a hallmark of stroke. Neural Ca2+ homeostasis involves regulation by the Na+/Ca2+ exchanger (NCX). Previous data published by our group showed that a product of the enzymatic depolymerization of heparin by heparinase, the unsaturated trisulfated disaccharide (TD; ΔU, 2S-GlcNS, 6S), can accelerate Na+/Ca2+ exchange via NCX, in hepatocytes and aorta vascular smooth muscle cells. Thus, the objective of this work was to verify whether TD could act as a neuroprotective agent able to prevent neuronal cell death by reducing [Ca2+]i. Pretreatment of N2a cells with TD reduced [Ca2+]i rise induced by thapsigargin and increased cell viability under [Ca2+]I overload conditions and in hypoxia. Using a murine model of stroke, we observed that pretreatment with TD decreased cerebral infarct volume and cell death. However, when mice received KB-R7943, an NCX blocker, the neuroprotective effect of TD was abolished, strongly suggesting that this neuroprotection requires a functional NCX to happen. Thus, we propose TD-NCX as a new therapeutic axis for the prevention of neuronal death induced by [Ca2+]i overload.

A new function for Prokineticin 2: Recruitment of SVZ-derived neuroblasts to the injured cortex in a mouse model of traumatic brain injury.

Traumatic brain injury is an important cause of global morbidity and mortality. After an initial injury, there is a cascade of cellular and molecular events that ultimately lead to cell death. Therapies aim to both counteract these mechanisms and replenish the lost cell population in order to improve recovery. The adult mammal brain has at least two neurogenic regions that maintain physiological functions: the subgranular zone of the dentate gyrus in the hippocampus, which produces neurons that integrate locally, and the subventricular zone (SVZ) adjacent to the lateral ventricles, which produces neuroblasts that migrate through the rostral migratory stream (RMS) to the olfactory bulbs. Brain injuries, as well as neurodegenerative diseases, induce the SVZ to respond by increasing cell proliferation and migration to the injured areas. Here we report that cells migrate from the SVZ and RMS to the injured cortex after traumatic brain injury in mice, and that the physiological RMS migration is not impaired. We also show that Prokineticin 2 (PROK2), a chemokine important for the olfactory bulb neurogenesis, expressed exclusively by cortical microglia in the cortex as early as 24 h after injury. We then show that administration of a PROK2 receptor antagonist decreases the number of SVZ cells that reach the injured cortex, while injection of recombinant PROK2 into the cortex of uninjured mice attracts SVZ cells. We also demonstrate that cells expressing PROK2 in vitro directionally attract SVZ cells. These data suggest that PROK2 could be utilized in regeneration efforts for the acutely injured mammalian cortex.

Rotary jet-spun porous microfibers as scaffolds for stem cells delivery to central nervous system injury.

Stem cell transplantation is a promising strategy to treat brain injuries. However, cell-based therapies are limited because poor local cell engraftment. Here, we present a polylactic acid (PLA) scaffold to support mesenchymal stem cells (MSCs) delivery in stroke. We isolated bone marrow MSCs from adult C57/Bl6 mice, cultured them on PLA polymeric rough microfibrous (PRM) scaffolds obtained by rotary jet spinning, and transplanted over the brains of adult C57/Bl6 mice, carrying thermocoagulation-induced cortical stroke. No inflammatory response to PRM was found. MSCs transplantation significantly reduced the area of the lesion and PRM delivery increased MSCs retention at the injury site. In addition, PRM upregulated α6-integrin and CXCL12 production, which may be the cause for greater cell retention at the lesion site and may provide additional benefit to MSCs transplantation procedures. We conclude that PRM scaffolds offer a promising new system to deliver stem cells to injured areas of the brain.

Polypyrrole increases branching and neurite extension by Neuro2A cells on PBAT ultrathin fibers.

We present a methodology for production and application of electrospun hybrid materials containing commercial polyester (poly (butylene adipate-co-terephthalate; PBAT), and a conductive polymer (polypyrrole; PPy) as scaffold for neuronal growth and differentiation. The physical-chemical properties of the scaffolds and optimization of the electrospinning parameters are presented. The electrospun scaffolds are biocompatible and allow proper adhesion and spread of mesenchymal stem cells (MSCs). Fibers produced with PBAT with or without PPy were used as scaffold for Neuro2a mouse neuroblastoma cells adhesion and differentiation. Neuro2a adhered to PBAT and PBAT/PPy2% scaffolds without laminin coating. However, Neuro2a failed to differentiate in PBAT when stimulated by treatment with retinoic acid (RA), but differentiated in PBAT/PPy2% fibers. We hypothesize that PBAT hydrophobicity inhibited proper spreading and further differentiation, and inhibition was overcome by coating the PBAT fibers with laminin. We conclude that fibers produced with the combination of PBAT and PPy can support neuronal differentiation.

Bioactivity behaviour of nano-hydroxyapatite/freestanding aligned carbon nanotube oxide composite.

Bioactive and low cytotoxic three dimensional nano-hydroxyapatite (nHAp) and aligned carbon nanotube oxide (a-CNTO) composite has been investigated. First, freestanding aligned carbon nanotubes porous scaffold was prepared by large-scale thermal chemical vapour deposition and functionalized by oxygen plasma treatment, forming a-CNTO. The a-CNTO was covered with plate-like nHAp crystals prepared by in situ electrodeposition techniques, forming nHAp/a-CNTO composite. After that nHAp/a-CNTO composite was immersed in simulated body fluid for composite consolidation. This novel nanobiomaterial promotes mesenchymal stem cell adhesion with the active formation of membrane projections, cell monolayer formation and high cell viability.

Mechanobiological Modulation of In Vitro Astrocyte Reactivity Using Variable Gel Stiffness.

After traumatic brain injury, the brain extracellular matrix undergoes structural rearrangement due to changes in matrix composition, activation of proteases, and deposition of chondroitin sulfate proteoglycans by reactive astrocytes to produce the glial scar. These changes lead to a softening of the tissue, where the stiffness of the contusion "core" and peripheral "pericontusional" regions becomes softer than that of healthy tissue. Pioneering mechanotransduction studies have shown that soft substrates upregulate intermediate filament proteins in reactive astrocytes; however, many other aspects of astrocyte biology remain unclear. Here, we developed a platform for the culture of cortical astrocytes using polyacrylamide (PA) gels of varying stiffness (measured in Pascal; Pa) to mimic injury-related regions in order to investigate the effects of tissue stiffness on astrocyte reactivity and morphology. Our results show that substrate stiffness influences astrocyte phenotype; soft 300 Pa substrates led to increased GFAP immunoreactivity, proliferation, and complexity of processes. Intermediate 800 Pa substrates increased Aggrecan+, Brevican+, and Neurocan+ astrocytes. The stiffest 1 kPa substrates led to astrocytes with basal morphologies, similar to a physiological state. These results advance our understanding of astrocyte mechanotransduction processes and provide evidence of how substrates with engineered stiffness can mimic the injury microenvironment.

High Concentrations of Cannabidiol Induce Neurotoxicity in Neurosphere Culture System.

Recent studies have demonstrated that cannabinoids are potentially effective in the treatment of various neurological conditions, and cannabidiol (CBD), one of the most studied compounds, has been proposed as a non-toxic option. However, the adverse effects of CBD on neurodevelopmental processes have rarely been studied in cell culture systems. To better understand CBD's influence on neurodevelopment, we exposed neural progenitor cells (NPCs) to different concentrations of CBD (1 µM, 5 µM, and 10 µM). We assessed the morphology, migration, differentiation, cell death, and gene expression in 2D and 3D bioprinted models to stimulate physiological conditions more effectively. Our results showed that CBD was more toxic at higher concentrations (5 µM and 10 µM) and affected the viability of NPCs than at lower concentrations (1 µM), in both 2D and 3D models. Moreover, our study revealed that higher concentrations of CBD drastically reduced the size of neurospheres and the number of NPCs within neurospheres, impaired the morphology and mobility of neurons and astrocytes after differentiation, and reduced neurite sprouting. Interestingly, we also found that CBD alters cellular metabolism by influencing the expression of glycolytic and ß-oxidative enzymes in the early and late stages of metabolic pathways. Therefore, our study demonstrated that higher concentrations of CBD promote important changes in cellular functions that are crucial during CNS development.

Posttraumatic epilepsy: Integrating clinical, inflammatory, and genetic profiles in traumatic brain injury patients.

OBJECTIVE: This study aims to assess the clinical, inflammatory, and genetic profiles of traumatic brain injury (TBI) patients over a 2-year follow-up period, focusing on the development of posttraumatic epilepsy (PTE). METHODS: Fifty-nine patients with acute TBI were recruited in the emergency unit of a hospital in Brazil. Clinical data and blood samples were collected after 10 days of hospitalization for posterior genetic profile (Apolipoprotein E- ApoE and Glutamic Acid Descarboxylase-GAD sequencing) analyses. A subset of 19 patients were assessed for cytokine markers (mRNA expression). The development of PTE was investigated for two years following TBI. Statistical analyses including univariate analysis, multiple correspondence analysis, and Mann-Whitney test were performed. RESULTS: Analysis revealed an association between severe TBI and requirement for neurosurgery and polytrauma (p<0.05), as well as the development of PTE over a two-year follow-up period (p<0.05). Multiple correspondence analysis identified two distinct profiles associated with PTE and Non-PTE outcomes. The PTE profile showed a higher prevalence of the ApoE genotype E3/E3 and GAD1 SNP (rs769391) genotype AA in our study, while the Non-PTE profile showed a higher presence of E3/E4. mRNA expression analysis demonstrated acute elevated levels of TNF-α in the PTE group as compared to Non-PTE patients (6.70±1.53 vs 5.31 ±0.33, p<0.01). SIGNIFICANCE: Our findings underscore the multifactorial nature of aspects potentially contributing to PTE. It is unlikely that any single factor might in isolation have a strong causative influence over the development of epilepsy after TBI. Our results provide a suggestion of potential clustering that might be relevant as prognostic factors for PTE.

Generation and characterization of cortical organoids from iPSC-derived dental pulp stem cells using traditional and innovative approaches.

Cortical organoids derived from human induced pluripotent stem cells (hiPSCs) represent a powerful in vitro experimental system to investigate human brain development and disease, often inaccessible to direct experimentation. However, despite steady progress in organoid technology, several limitations remain, including high cost and variability, use of hiPSCs derived from tissues harvested invasively, unexplored three-dimensional (3D) structural features and neuronal connectivity. Here, using a cost-effective and reproducible protocol as well as conventional two-dimensional (2D) immunostaining, we show that cortical organoids generated from hiPSCs obtained by reprogramming stem cells from human exfoliated deciduous teeth (SHED) recapitulate key aspects of human corticogenesis, such as polarized organization of neural progenitor zones with the presence of outer radial glial stem cells, and differentiation of superficial- and deep-layer cortical neurons and glial cells. We also show that 3D bioprinting and magnetic resonance imaging of intact cortical organoids are alternative and complementary approaches to unravel critical features of the 3D architecture of organoids. Finally, extracellular electrical recordings in whole organoids showed functional neuronal networks. Together, our findings suggest that SHED-derived cortical organoids constitute an attractive model of human neurodevelopment, and support the notion that a combination of 2D and 3D techniques to analyze organoid structure and function may help improve this promising technology.

Protocol for designing and bioprinting multi-layered constructs to reconstruct an endothelial-epithelial 3D model.

3D bioprinting has opened new possibilities and elevated tissue engineering complexity. Here, we present a protocol to design a 3D model with two cell lineage layers (A549 and HUVEC) to recreate multi-cell constructs. We describe the steps for slicing the constructs, handling hydrogels, and detailing the bioprinting setup. These 3D-bioprinted constructs can be adapted to various cell models-from primary cell cultures to commercial cell lines and induced pluripotent stem cells (IPCs)-and applications, including drug screening and disease modeling. For complete details on the use and execution of this protocol, please refer to Cruz et al.1.

Sleep deprivation modulates APOE and LDL receptor-related protein 1 through thyroid hormone T4 and impairs Aß clearance in hippocampus of rats.

Alzheimer's disease is the most common form of dementia. One of its pathological hallmarks is Aß accumulation, which is influenced by APOE genotype and expression, as well as by sleep homeostasis. However, conflicting mechanisms for APOE roles in Aß clearance have been reported, and the relationship between APOE and sleep also remains unclear. In this study, we aimed to investigate how hormonal alteration caused by sleep deprivation affects APOE and its receptors in rats, and to evaluate the role of different cell types in Aß clearance. Paradoxical sleep deprivation for 96 h increased Aß level in hippocampus with concomitant reduction of APOE and LRP1 at the time point within the resting period. Sleep deprivation also significantly reduced T4 levels in both active and resting times. To evaluate the effect of T4 variation, C6 glial cells and primary brain endothelial cells were treated with T4. High T4 level (300 ng/mL) increased APOE, but reduced LRP1 and LDL-R in C6 cells, while in primary endothelial cells, LDL-R levels were increased. Treatment of C6 cells with exogenous APOE reduced LRP1 and Aß uptake. These results suggest that T4 modulates LRP1 and LDL-R in both cell types, but in the opposite manner, thus, sleep deprivation might modify the ratio of the receptors in blood-brain barrier and glial cells by altering T4 levels. Considering that LRP1 and LDL-R are important for Aß clearance, sleep deprivation might also affect the degree of participation of glia in Aß clearance, and consequently, turnover of Aß in the brain.

A Gelatin Methacrylate-Based Hydrogel as a Potential Bioink for 3D Bioprinting and Neuronal Differentiation.

Neuronal loss is the ultimate pathophysiologic event in central nervous system (CNS) diseases and replacing these neurons is one of the most significant challenges in regenerative medicine. Providing a suitable microenvironment for new neuron engraftment, proliferation, and synapse formation is a primary goal for 3D bioprinting. Among the various biomaterials, gelatin methacrylate (GelMA) stands out due to its Arg-Gly-Asp (RGD) domains, which assure its biocompatibility and degradation under physiological conditions. This work aimed to produce different GelMA-based bioink compositions, verify their mechanical and biological properties, and evaluate their ability to support neurogenesis. We evaluated four different GelMA-based bioink compositions; however, when it came to their biological properties, incorporating extracellular matrix components, such as GeltrexTM, was essential to ensure human neuroprogenitor cell viability. Finally, GeltrexTM: 8% GelMA (1:1) bioink efficiently maintained human neuroprogenitor cell stemness and supported neuronal differentiation. Interestingly, this bioink composition provides a suitable environment for murine astrocytes to de-differentiate into neural stem cells and give rise to MAP2-positive cells.

Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior.

BACKGROUND: Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity. METHODS: MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses. RESULTS: After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months. CONCLUSIONS: These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.

Self-organization of in vitro neuronal assemblies drives to complex network topology.

Activity-dependent self-organization plays an important role in the formation of specific and stereotyped connectivity patterns in neural circuits. By combining neuronal cultures, and tools with approaches from network neuroscience and information theory, we can study how complex network topology emerges from local neuronal interactions. We constructed effective connectivity networks using a transfer entropy analysis of spike trains recorded from rat embryo dissociated hippocampal neuron cultures between 6 and 35 days in vitro to investigate how the topology evolves during maturation. The methodology for constructing the networks considered the synapse delay and addressed the influence of firing rate and population bursts as well as spurious effects on the inference of connections. We found that the number of links in the networks grew over the course of development, shifting from a segregated to a more integrated architecture. As part of this progression, three significant aspects of complex network topology emerged. In agreement with previous in silico and in vitro studies, a small-world architecture was detected, largely due to strong clustering among neurons. Additionally, the networks developed in a modular topology, with most modules comprising nearby neurons. Finally, highly active neurons acquired topological characteristics that made them important nodes to the network and integrators of modules. These findings leverage new insights into how neuronal effective network topology relates to neuronal assembly self-organization mechanisms.

Simple and efficient protocol to isolate and culture brain microvascular endothelial cells from newborn mice.

The neurovascular unit (NVU) is a multicellular structure comprising of neurons, glial cells, and non-neural cells, and it is supported by a specialized extracellular matrix, the basal lamina. Astrocytes, brain microvascular endothelial cells (BMECs), pericytes, and smooth muscle cells constitute the blood-brain barrier (BBB). BMECs have a mesodermal origin and invade the nervous system early in neural tube development, forming the BBB anatomical core. BMECs are connected by adherent junction complexes composed of integral membrane and cytoplasmic proteins. In vivo and in vitro studies have shown that, given the proximity and relationship with neural cells, BMECs acquire a unique gene expression profile, proteome, and specific mechanical and physical properties compared to endothelial cells from the general vasculature. BMECs are fundamental in maintaining brain homeostasis by regulating transcellular and paracellular transport of fluids, molecules, and cells. Therefore, it is essential to gain in-depth knowledge of the dynamic cellular structure of the cells in the NVU and their interactions with health and disease. Here we describe a significantly improved and simplified protocol using C57BL/6 newborn mice at postnatal day 1 (PND1) to isolate, purify, and culture BMECs monolayers in two different substrates (glass coverslips and transwell culture inserts). In vitro characterization and validation of the BMEC primary culture monolayers seeded on glass or insert included light microscopy, immunolabeling, and gene expression profile. Transendothelial electrical resistance (TEER) measurement and diffusion test were used as functional assays for adherent junction complexes and integrity and permeability of BMECs monolayers. The protocol presented here for the isolation and culture of BMECs is more straightforward than previously published protocols and yields a high number of purified cells. Finally, we tested BMECs function using the oxygen-glucose deprivation (OGD) model of hypoxia. This protocol may be suitable as a bioscaffold for secondary cell seeding allowing the study and better understanding of the NVU.

Biomimetic Nanovesicles-Sources, Design, Production Methods, and Applications.

Despite all the progress in the field of liposomes and nanoparticles for applications as drug and gene delivery systems, the specific targeting and immune system escape capabilities of these systems are still limited. Biomimetic nanovesicles emerged as a strategy to overcome these and other limitations associated with synthetic carriers, such as short circulation time, cytotoxicity, and difficulty in crossing biological barriers, since many of the desirable abilities of drug delivery systems are innate characteristics of biological vesicles. Thus, the question arises: would biomimetic nanovesicles be responsible for addressing these advances? It is currently known that biomimetic nanovesicles (BNV) can combine the intrinsic advantages of natural materials with the well-known production methods and controllability of synthetic systems. Besides, the development of the biotechnology and nanotechnology fields has provided a better understanding of the functionalities of biological vesicles and the means for the design and production of biomimetic nanovesicles (BNV). Based on this, this work will focus on tracking the main research on biomimetic nanovesicles (BNV) applied as drug and gene delivery systems, and for vaccines applications. In addition, it will describe the different sources of natural vesicles, the technical perspectives on obtaining them, and the possibility of their hybridization with synthetic liposomes.

3D Bioprinted Neural-Like Tissue as a Platform to Study Neurotropism of Mouse-Adapted SARS-CoV-2.

The effects of neuroinvasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) become clinically relevant due to the numerous neurological symptoms observed in Corona Virus Disease 2019 (COVID-19) patients during infection and post-COVID syndrome or long COVID. This study reports the biofabrication of a 3D bioprinted neural-like tissue as a proof-of-concept platform for a more representative study of SARS-CoV-2 brain infection. Bioink is optimized regarding its biophysical properties and is mixed with murine neural cells to construct a 3D model of COVID-19 infection. Aiming to increase the specificity to murine cells, SARS-CoV-2 is mouse-adapted (MA-SARS-CoV-2) in vitro, in a protocol first reported here. MA-SARS-CoV-2 reveals mutations located at the Orf1a and Orf3a domains and is evolutionarily closer to the original Wuhan SARS-CoV-2 strain than SARS-CoV-2 used for adaptation. Remarkably, MA-SARS-CoV-2 shows high specificity to murine cells, which present distinct responses when cultured in 2D and 3D systems, regarding cell morphology, neuroinflammation, and virus titration. MA-SARS-CoV-2 represents a valuable tool in studies using animal models, and the 3D neural-like tissue serves as a powerful in vitro platform for modeling brain infection, contributing to the development of antivirals and new treatments for COVID-19.