Early Alzheimer's disease pathology in human cortex involves transient cell states.

Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the early cortical amyloid response-were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with ß-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.

Reduced binding of apoE4 to complement factor H promotes amyloid-ß oligomerization and neuroinflammation.

The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late-onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform-specific binding of apoE to FH alters Aß1-42-mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aß1-42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement-resistant oligomers with apoE/Aß1-42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aß1-42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aß plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.

Functional Characterization of Human Pluripotent Stem Cell-Derived Models of the Brain with Microelectrode Arrays.

Human pluripotent stem cell (hPSC)-derived neuron cultures have emerged as models of electrical activity in the human brain. Microelectrode arrays (MEAs) measure changes in the extracellular electric potential of cell cultures or tissues and enable the recording of neuronal network activity. MEAs have been applied to both human subjects and hPSC-derived brain models. Here, we review the literature on the functional characterization of hPSC-derived two- and three-dimensional brain models with MEAs and examine their network function in physiological and pathological contexts. We also summarize MEA results from the human brain and compare them to the literature on MEA recordings of hPSC-derived brain models. MEA recordings have shown network activity in two-dimensional hPSC-derived brain models that is comparable to the human brain and revealed pathology-associated changes in disease models. Three-dimensional hPSC-derived models such as brain organoids possess a more relevant microenvironment, tissue architecture and potential for modeling the network activity with more complexity than two-dimensional models. hPSC-derived brain models recapitulate many aspects of network function in the human brain and provide valid disease models, but certain advancements in differentiation methods, bioengineering and available MEA technology are needed for these approaches to reach their full potential.

Microglia-like Cells Promote Neuronal Functions in Cerebral Organoids.

Human cerebral organoids, derived from induced pluripotent stem cells, offer a unique in vitro research window to the development of the cerebral cortex. However, a key player in the developing brain, the microglia, do not natively emerge in cerebral organoids. Here we show that erythromyeloid progenitors (EMPs), differentiated from induced pluripotent stem cells, migrate to cerebral organoids, and mature into microglia-like cells and interact with synaptic material. Patch-clamp electrophysiological recordings show that the microglia-like population supported the emergence of more mature and diversified neuronal phenotypes displaying repetitive firing of action potentials, low-threshold spikes and synaptic activity, while multielectrode array recordings revealed spontaneous bursting activity and increased power of gamma-band oscillations upon pharmacological challenge with NMDA. To conclude, microglia-like cells within the organoids promote neuronal and network maturation and recapitulate some aspects of microglia-neuron co-development in vivo, indicating that cerebral organoids could be a useful biorealistic human in vitro platform for studying microglia-neuron interactions.

A modular brain-on-a-chip for modelling epileptic seizures with functionally connected human neuronal networks.

Epilepsies are a group of neurological disorders characterised by recurrent epileptic seizures. Seizures, defined as abnormal transient discharges of neuronal activity, can affect the entire brain circuitry or remain more focal in the specific brain regions and neuronal networks. Human pluripotent stem cell (hPSC)-derived neurons are a promising option for modelling epilepsies, but as such, they do not model groups of connected neuronal networks or focal seizures. Our solution is a Modular Platform for Epilepsy Modelling In Vitro (MEMO), a lab-on-chip device, in which three hPSC-derived networks are separated by a novel microfluidic cell culture device that allows controlled network-to-network axonal connections through microtunnels. In this study, we show that the neuronal networks formed a functional circuitry that was successfully cultured in MEMO for up to 98 days. The spontaneous neuronal network activities were monitored with an integrated custom-made microelectrode array (MEA). The networks developed spontaneous burst activity that was synchronous both within and between the axonally connected networks, i.e. mimicking both local and circuitry functionality of the brain. A convulsant, kainic acid, increased bursts only in the specifically treated networks. The activity reduction by an anticonvulsant, phenytoin, was also localised to treated networks. Therefore, modelling focal seizures in human neuronal networks is now possible with the developed chip.

Microelectrode Array With Transparent ALD TiN Electrodes.

Low noise platinum black or sputtered titanium nitride (TiN) microelectrodes are typically used for recording electrical activity of neuronal or cardiac cell cultures. Opaque electrodes and tracks, however, hinder the visibility of the cells when imaged with inverted microscope, which is the standard method of imaging cells plated on microelectrode array (MEA). Even though transparent indium tin oxide (ITO) electrodes exist, they cannot compete in impedance and noise performance with above-mentioned opaque counterparts. In this work, we propose atomic layer deposition (ALD) as the method to deposit TiN electrodes and tracks which are thin enough (25-65 nm) to be transparent (transmission ∼18-45%), but still benefit from the columnar structure of TiN, which is the key element to decrease noise and impedance of the electrodes. For ALD TiN electrodes (diameter 30 µm) impedances from 510 to 590 kΩ were measured at 1 kHz, which is less than the impedance of bare ITO electrodes. Human induced pluripotent stem cell (hiPSC)-derived cortical neurons were cultured on the ALD TiN MEAs for 14 days without observing any biocompatibility issues, and spontaneous electrical activity of the neurons was recorded successfully. The results show that transparent ALD TiN film is a suitable electrode material for producing functional MEAs.

Effect of prolonged differentiation on functional maturation of human pluripotent stem cell-derived neuronal cultures.

Long-term neural differentiation of human pluripotent stem cells (hPSCs) is associated with enhanced neuronal maturation, which is a necessity for creation of representative in vitro models. It also induces neurogenic-to-gliogenic fate switch, increasing proportion of endogenous astrocytes formed from the common neural progenitors. However, the significance of prolonged differentiation on the neural cell type composition and functional development of hPSC-derived neuronal cells has not been well characterized. Here, we studied two hPSC lines, both of which initially showed good neuronal differentiation capacity. However, the propensity for endogenous astrogenesis and maturation state after extended differentiation varied. Live cell calcium imaging revealed that prolonged differentiation facilitated maturation of GABAergic signaling. According to extracellular recordings with microelectrode array (MEA), neuronal activity was limited to fewer areas of the culture, which expressed more frequent burst activity. Efficient maturation after prolonged differentiation also promoted organization of spontaneous activity by burst compaction. These results suggest that although prolonged neural differentiation can be challenging, it has beneficial effect on functional maturation, which can also improve transition to different neural in vitro models and applications.

Optimised PDMS Tunnel Devices on MEAs Increase the Probability of Detecting Electrical Activity from Human Stem Cell-Derived Neuronal Networks.

Measurement of the activity of human pluripotent stem cell (hPSC)-derived neuronal networks with microelectrode arrays (MEAs) plays an important role in functional in vitro brain modelling and in neurotoxicological screening. The previously reported hPSC-derived neuronal networks do not, however, exhibit repeatable, stable functional network characteristics similar to rodent cortical cultures, making the interpretation of results difficult. In earlier studies, microtunnels have been used both to control and guide cell growth and amplify the axonal signals of rodent neurons. The aim of the current study was to develop tunnel devices that would facilitate signalling and/or signal detection in entire hPSC-derived neuronal networks containing not only axons, but also somata and dendrites. Therefore, MEA-compatible polydimethylsiloxane (PDMS) tunnel devices with 8 different dimensions were created. The hPSC-derived neurons were cultured in the tunnel devices on MEAs, and the spontaneous electrical activity of the networks was measured for 5 weeks. Although the tunnel devices improved the signal-to-noise ratio only by 1.3-fold at best, they significantly increased the percentage of electrodes detecting neuronal activity (52-100%) compared with the controls (27%). Significantly higher spike and burst counts were also obtained using the tunnel devices. Neuronal networks inside the tunnels were amenable to pharmacological manipulation. The results suggest that tunnel devices encompassing the entire neuronal network can increase the measured spontaneous activity in hPSC-derived neuronal networks on MEAs. Therefore, they can increase the efficiency of functional studies of hPSC-derived networks on MEAs.

Effects of exogenous alpha-synuclein on stimulated dopamine overflow in dorsal striatum.

Alpha-synuclein (α-syn) is mainly a presynaptic protein that has been implicated in Parkinson's disease and various other neurodegenerative disorders. Evidence obtained in knockout mice suggests that α-syn controls plasticity of dopamine (DA) overflow in presynaptic terminals. It is also believed that α-syn spreads and may seed its aggregates from cell to cell. The effects of exogenously applied α-syn on dopaminergic neurotransmission have not been studied. We addressed this issue by microinjecting human α-syn protein into the dorsal striatum of wild-type and α-syn knockout mice and monitoring stimulated DA overflow with constant potential amperometry. The evoked DA overflow was decreased in knockout mice six days after α-syn microinjection. The maximal velocity of DA re-uptake was reduced in both genotypes. Similar results were not seen when the effects of microinjected α-syn were studied immediately after the treatment, but instead there was a trend toward an increase in both stimulated DA overflow and maximal velocity of DA re-uptake. We conclude that locally applied human α-syn affects DA overflow and the effects depend on the presence of endogenous α-syn.

Neuromuscular pathology in mice lacking alpha-synuclein.

This work was undertaken in order to study the possible role of alpha-synuclein in the function of the neuro-muscular junction in skeletal muscles. Repeated stimulation of skeletal muscle motor neurons revealed signs of neuromuscular pathology in alpha-synuclein null mutated (C57Bl/6JOlaHsd) and knockout (B6;129X1-Snca(tm1Rosl)/J) mice. This stimulation produced repetitive compound muscle action potentials in both lines of alpha-synuclein deficient mice. Muscle strength and muscle coordination during ambulation were unaffected, though motor learning was slower in alpha-synuclein deficient mice in the Rotarod test. We conclude that alpha-synuclein may play a role in acetylcholine compartmentalization at the neuromuscular junction, and in the fine control of activity of skeletal muscles.

Stimulated dopamine overflow and alpha-synuclein expression in the nucleus accumbens core distinguish rats bred for differential ethanol preference.

The key neurochemical systems and structures involved in the predisposition to substance abuse and preference to ethanol (EtOH) are not known in detail but clearly dopamine (DA) is an important modulator of addiction. Recent data indicate that alpha-synuclein (alpha-syn), a pre-synaptic protein, plays a role in regulation of DA release from the pre-synaptic terminals in striatum and the expression of this protein is different after drug abuse or following abstinence. In the present work, we analysed stimulated DA overflow in the dorsal and ventral striatum in EtOH naïve alko alchohol (AA) and alko non-alchohol (ANA) rats selected for more than 100 generations for their differential EtOH preference. In the same structures, we studied the expression of alpha-syn using western blotting. AA rats, in comparison with ANA rats, showed a marked reduction of stimulated peak DA overflow and higher levels of alpha-syn in the nucleus accumbens core. In the same structure, DA re-uptake was increased in AA rats in comparison with ANA rats. The effects of EtOH at low (0.1 g/kg) and higher (3 mg/kg) doses on DA overflow measured in the nucleus accumbens shell were similar in both lines. These results indicate that high expression of alpha-syn may contribute to the reduced DA overflow and the possible activation of re-uptake in the nucleus accumbens core of AA rats in comparison with ANA rats.