We are accumulating evidence that intestinal microflora, collectively named gut microbiota, can alter brain pathophysiology, but researchers have just begun to discover the mechanisms of this bidirectional connection (often referred to as microbiota-gut-brain axis, MGBA). The most noticeable hypothesis for a pathological action of gut microbiota on the brain is based on microbial release of soluble neurotransmitters, hormones, immune molecules and neuroactive metabolites, but this complex scenario requires reliable and controllable tools for its causal demonstration. Thanks to three-dimensional (3D) cultures and microfluidics, engineered in vitro models could improve the scientific knowledge in this field, also from a therapeutic perspective. This review briefly retraces the main discoveries linking the activity of gut microbiota to prevalent brain neurodegenerative disorders, and then provides a deep insight into the state-of-the-art for in vitro modeling of the brain and the blood-brain barrier (BBB), two key players of the MGBA. Several brain and BBB microfluidic devices have already been developed to implement organ-on-a-chip solutions, but some limitations still exist. Future developments of organ-on-a-chip tools to model the MGBA will require an interdisciplinary approach and the synergy with cutting-edge technologies (for instance, bioprinting) to achieve multi-organ platforms and support basic research, also for the development of new therapies against neurodegenerative diseases. Keywords: microbiota-gut-brain axis, neurodegenerative diseases, in vitro modeling, microfluidics, brain, bloodbrain barrier THE MICROBIOTA-GUT-BRAIN AXIS AND ITS INVOLVEMENT IN NEUROLOGICAL DISEASES Research on microbiota is rooted in Antonie van Leeuwenhoek's pioneering work (XVII century) (Bardell, 1983), but in recent years it has aroused a growing interest. In particular, the gut microbiota has strongly emerged as a key player both in physiological and pathological conditions (Jia et al., 2008). The gut microbiota is a complex and dynamic population of tens of trillions of microbes residing in the gastrointestinal (GI) tract, with a mutualistic relationship with the host. Bacterial genera Brain disease References 5-Hydroxytryptamine (5-TH)

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The steady increase in life-expectancy of world population, coupled to many genetic and environmental factors (for instance, pre- and post-natal exposures to environmental neurotoxins), predispose to the onset of neurodegenerative diseases, whose prevalence is expected to increase dramatically in the next years. Recent studies have proposed links between the gut microbiota and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Human body is a complex structure where bacterial and human cells are almost equal in numbers, and most microbes are metabolically active in the gut, where they potentially influence other target organs, including the brain. The role of gut microbiota in the development and pathophysiology of the human brain is an area of growing interest for the scientific community. Several microbial-derived neurochemicals involved in the gut-microbiota-brain crosstalk seem implicated in the biological and physiological basis of neurodevelopment and neurodegeneration. Evidence supporting these connections has come from model systems, but there are still unsolved issues due to several limitations of available research tools. New technologies are recently born to help understanding the causative role of gut microbes in neurodegeneration. This review aims to make an overview of recent advances in the study of the microbiota-gut-brain axis in the field of neurodegenerative disorders by: (a) identifying specific microbial pathological signaling pathways; (b) characterizing new, advanced engineered tools to study the interactions between human cells and gut bacteria.

Recent findings have highlighted an interconnection between intestinal microbiota and the brain, referred to as microbiota–gut–brain axis, and suggested that alterations in microbiota composition might affect brain functioning, also in Alzheimer’s disease. To investigate microbiota–gut–brain axis biochemical pathways, in this work we developed an innovative device to be used as modular unit in an engineered multi-organ-on-a-chip platform recapitulating in vitro the main players of the microbiota–gut–brain axis, and an innovative three-dimensional model of brain cells based on collagen/hyaluronic acid or collagen/poly(ethylene glycol) semi-interpenetrating polymer networks and β-amyloid precursor protein-Swedish mutant-expressing H4 cells, to simulate the pathological scenario of Alzheimer’s disease. We set up the culturing conditions, assessed cell response, scaled down the three-dimensional models to be hosted in the organ-on-a-chip device, and cultured them both in static and in dynamic conditions. The results suggest that the device and three-dimensional models are exploitable for advanced engineered models representing brain features also in Alzheimer’s disease scenario.

Neurodegenerative disorders such as Parkinson's disease (PD) have no effective therapies. However, many promising drugs are precluded from clinical trials because of their poor brain availability. The chaperone protein Hsp70 has been reported to be effective in PD models, but its brain targeting is challenging. We developed a novel brain Hsp70 delivery system using injectable, biocompatible, and biodegradable semi-interpenetrating polymer networks of collagen (COLL) and low-molecular-weight hyaluronic acid (LMW HA) structured with gelatin particles. We produced human recombinant Hsp70-1A fused with the cell-penetrating peptide Tat (Tat-Hsp70) that was neuroprotective in vitro against the dopaminergic toxin 6-hydroxydopamine (6-OHDA). We assessed Tat-Hsp70 release from the selected COLL-LMW HA composites in vitro, observing a 95% release of loaded protein after 96 h. The release kinetics FITTED the Korsmeyer-Peppas model (regression coefficient 0.98) and the released Tat-Hsp70 remained neuroprotective for SH-SY5Y cells. Magnetic resonance imaging revealed that COLL-LMW HA composites lasted at least 96 h at the brain level, and in vivo Tat-Hsp70 release studies indicated that hydrogel presence is pivotal for a spatially focused neuroprotective effect. In an in vivo model of dopaminergic degeneration, Tat-Hsp70-loaded composites conveyed neuroprotection at both the behavioral and dopaminergic neuronal levels against the striatal injection of 6-OHDA. After the injection of Tat-Hsp70-loaded composites, mice showed a transient inflammatory response, with a decrease in GFAP and CD11b immunostaining after 7 days. Our delivery system enabled the effective brain release of Tat-Hsp70 and is ready for further improvements.