Integrating deep learning and unbiased automated high-content screening to identify complex disease signatures in human fibroblasts.

Drug discovery for diseases such as Parkinson's disease are impeded by the lack of screenable cellular phenotypes. We present an unbiased phenotypic profiling platform that combines automated cell culture, high-content imaging, Cell Painting, and deep learning. We applied this platform to primary fibroblasts from 91 Parkinson's disease patients and matched healthy controls, creating the largest publicly available Cell Painting image dataset to date at 48 terabytes. We use fixed weights from a convolutional deep neural network trained on ImageNet to generate deep embeddings from each image and train machine learning models to detect morphological disease phenotypes. Our platform's robustness and sensitivity allow the detection of individual-specific variation with high fidelity across batches and plate layouts. Lastly, our models confidently separate LRRK2 and sporadic Parkinson's disease lines from healthy controls (receiver operating characteristic area under curve 0.79 (0.08 standard deviation)), supporting the capacity of this platform for complex disease modeling and drug screening applications.

Recent advances in genome editing of stem cells for drug discovery and therapeutic application.

Genome engineering technologies right from viral vector-mediated to protein-based editing- which include zinc finger nucleases, TALENs, and CRISPR/Cas systems-have been improved significantly. These technologies have facilitated drug discovery and have resulted in the development of potential curative therapies for many intractable diseases. They can efficiently correct genetic errors; however, these technologies have limitations, such as off-target effects and possible safety issues, which need to be considered when employing these techniques in humans. Significant efforts have been made to overcome these limitations and to accelerate the clinical implementation of these technologies. In this review, we focus on the recent technological advancements in genome engineering and their applications in stem cells to enable efficient discovery of drugs and treatment of intractable diseases.

Injury-induced alterations in CNS electrophysiology.

Mild to moderate cases of traumatic brain injury (TBI) are very common, but are not always associated with the overt pathophysiogical changes seen following severe trauma. While neuronal death has been considered to be a major factor, the pervasive memory, cognitive and motor function deficits suffered by many mild TBI patients do not always correlate with cell loss. Therefore, we assert that functional impairment may result from alterations in surviving neurons. Current research has begun to explore CNS synaptic circuits after traumatic injury. Here we review significant findings made using in vivo and in vitro models of TBI that provide mechanistic insight into injury-induced alterations in synaptic electrophysiology. In the hippocampus, research now suggests that TBI regionally alters the delicate balance between excitatory and inhibitory neurotransmission in surviving neurons, disrupting the normal functioning of synaptic circuits. In another approach, a simplified model of neuronal stretch injury in vitro, has been used to directly explore how injury impacts the physiology and cell biology of neurons in the absence of alterations in blood flow, blood brain barrier integrity, or oxygenation associated with in vivo models of brain injury. This chapter discusses how these two models alter excitatory and inhibitory synaptic transmission at the receptor, cellular and circuit levels and how these alterations contribute to cognitive impairment and a reduction in seizure threshold associated with human concussive brain injury.

Brain injury impairs dentate gyrus inhibitory efficacy.

Every 23 s, a person sustains a traumatic brain injury in the United States leaving many patients with substantial cognitive impairment and epilepsy. Injury-induced alterations in the hippocampus underpin many of these disturbances of neurological function. Abnormalities in the dentate gyrus are likely to play a major role in the observed pathophysiology because this subregion functions as a filter impeding excessive or aberrant activity from propagating further into the circuit and following experimental brain injury, the dentate gyrus becomes more excitable. Although alteration in excitation or inhibition could mediate this effect in the dentate gyrus, we show a key role played by an impairment of GABA(A)ergic inhibition. The efficacy of GABA(A)-mediated inhibition depends on a low [Cl-]i that is maintained by neuronal K-Cl co-transporter 2 (KCC2). Using fluid percussion injury (FPI) in the mouse, we demonstrate significant reductions in KCC2 protein and mRNA expression in the dentate gyrus that causes a depolarizing shift in GABA(A) reversal potential, due to impaired chloride clearance, resulting in reduced inhibitory efficiency. This study elucidates a novel mechanism underlying diminished dentate gyrus inhibitory efficacy and provides an innovative target for the development of potential therapeutics to restore the severe pathological consequences of traumatic brain injury.

Response of the contralateral hippocampus to lateral fluid percussion brain injury.

Traumatic brain injury is a leading cause of death and disability in the United States. Pathological examinations of humans and animal models after brain injury demonstrate hippocampal neuronal damage, which may contribute to cognitive impairments. Data from our laboratories have shown that, at 1 week after brain injury, mice possess significantly fewer neurons in all ipsilateral hippocampal subregions and a cognitive impairment. Since cognitive function is distributed across both cerebral hemispheres, the present paper explores the morphological and physiological response of the contralateral hippocampus to lateral brain injury. We analyzed the contralateral hippocampus using design-based stereology, Fluoro-Jade (FJ) histochemistry, and extracellular field recordings in mice at 7 and 30 days after lateral fluid percussion injury (FPI). At 7 days, all contralateral hippocampal subregions possess significantly fewer healthy neurons compared to sham-injured animals and demonstrate FJ-positive neuronal damage, but not at 30 days. Both the ipsilateral and contralateral dentate gyri demonstrate significantly increased excitability at 7 days post-injury, but only ipsilateral dentate gyrus hyperexcitability persists at 30 days compared to sham. In the contralateral hippocampus, the transient decrease in the number of healthy neurons, concomitant with FJ damage, and electrophysiological alterations establish a stunned period of cellular and circuit dysfunction. The return of healthy neuron number, absence of FJ damage, and sham level of excitability in the contralateral hippocampus suggest recovery of structure and function by 30 days after injury. The cognitive recovery observed after human traumatic brain injury may stem from a differential injury exposure and time course of recovery between homologous regions of the two hemispheres.