A spatially specified systems pharmacology therapy for axonal recovery after injury.

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axons using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Physiologically, drug treatment restored or preserved pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. Morphology experiments show that the four-drug combination protects axons or promotes axonal regrowth to the optic chiasm and beyond. We conclude that spatially targeted drug treatment is therapeutically relevant and can restore limited functional recovery.

Spinal cord injury regulates circular RNA expression in axons.

Introduction: Neurons transport mRNA and translational machinery to axons for local translation. After spinal cord injury (SCI), de novo translation is assumed to enable neurorepair. Knowledge of the identity of axonal mRNAs that participate in neurorepair after SCI is limited. We sought to identify and understand how axonal RNAs play a role in axonal regeneration. Methods: We obtained preparations enriched in axonal mRNAs from control and SCI rats by digesting spinal cord tissue with cold-active protease (CAP). The digested samples were then centrifuged to obtain a supernatant that was used to identify mRNA expression. We identified differentially expressed genes (DEGS) after SCI and mapped them to various biological processes. We validated the DEGs by RT-qPCR and RNA-scope. Results: The supernatant fraction was highly enriched for mRNA from axons. Using Gene Ontology, the second most significant pathway for all DEGs was axonogenesis. Among the DEGs was Rims2, which is predominately a circular RNA (circRNA) in the CNS. We show that Rims2 RNA within spinal cord axons is circular. We found an additional 200 putative circRNAs in the axonal-enriched fraction. Knockdown in primary rat cortical neurons of the RNA editing enzyme ADAR1, which inhibits formation of circRNAs, significantly increased axonal outgrowth and increased the expression of circRims2. Using Rims2 as a prototype we used Circular RNA Interactome to predict miRNAs that bind to circRims2 also bind to the 3'UTR of GAP-43, PTEN or CREB1, all known regulators of axonal outgrowth. Axonally-translated GAP-43 supports axonal elongation and we detect GAP-43 mRNA in the rat axons by RNAscope. Discussion: By enriching for axonal RNA, we detect SCI induced DEGs, including circRNA such as Rims2. Ablation of ADAR1, the enzyme that regulates circRNA formation, promotes axonal outgrowth of cortical neurons. We developed a pathway model using Circular RNA Interactome that indicates that Rims2 through miRNAs can regulate the axonal translation GAP-43 to regulate axonal regeneration. We conclude that axonal regulatory pathways will play a role in neurorepair.

Whole cell response to receptor stimulation involves many deep and distributed subcellular biochemical processes.

Neurite outgrowth is an integrated whole cell response triggered by the cannabinoid-1 receptor. We sought to identify the many different biochemical pathways that contribute to this whole cell response. To understand underlying mechanisms, we identified subcellular processes (SCPs) composed of one or more biochemical pathways and their interactions required for this response. Differentially expressed genes and proteins were obtained from bulk transcriptomics and proteomic analysis of extracts from cells stimulated with a cannabinoid-1 receptor agonist. We used these differentially expressed genes and proteins to build networks of interacting SCPs by combining the expression data with prior pathway knowledge. From these SCP networks, we identified additional genes that when ablated, experimentally validated the SCP involvement in neurite outgrowth. Our experiments and informatics modeling allowed us to identify diverse SCPs such as those involved in pyrimidine metabolism, lipid biosynthesis, and mRNA splicing and stability, along with more predictable SCPs such as membrane vesicle transport and microtubule dynamics. We find that SCPs required for neurite outgrowth are widely distributed among many biochemical pathways required for constitutive cellular functions, several of which are termed 'deep', since they are distal to signaling pathways and the key SCPs directly involved in extension of the neurite. In contrast, 'proximal' SCPs are involved in microtubule growth and membrane vesicle transport dynamics required for neurite outgrowth. From these bioinformatics and dynamical models based on experimental data, we conclude that receptor-mediated regulation of subcellular functions for neurite outgrowth is both distributed, that is, involves many different biochemical pathways, and deep.

Functional Effects of Cardiomyocyte Injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.

Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration.

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors, we have detected elevated levels of histone H3 in human CSF 24 h after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor Y-box-binding protein 1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C in vitro, and activated protein C treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS.

Physiology of cardiomyocyte injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System shows that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrate cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with SARS-CoV-2 in the presence of interleukins, with clinical findings, to investigate plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes, from healthy human subjects, with SARS-CoV-2 in the absence and presence of interleukins. We find that interleukin treatment and infection results in disorganization of myofibrils, extracellular release of troponin-I, and reduced and erratic beating. Although interleukins do not increase the extent, they increase the severity of viral infection of cardiomyocytes resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health system show that a significant portion of COVID-19 patients without prior history of heart disease, have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection can underlie the heart disease in COVID-19 patients.

Biomechanical Forces Regulate Gene Transcription During Stretch-Mediated Growth of Mammalian Neurons.

At birth, there are 100 billion neurons in the human brain, with functional neural circuits extending through the spine to the epidermis of the feet and toes. Following birth, limbs and vertebrae continue to grow by several orders of magnitude, forcing established axons to grow by up to 200 cm in length without motile growth cones. The leading regulatory paradigm suggests that biomechanical expansion of mitotic tissue exerts tensile force on integrated nervous tissue, which synchronizes ongoing growth of spanning axons. Here, we identify unique transcriptional changes in embryonic rat DRG and cortical neurons while the corresponding axons undergo physiological levels of controlled mechanical stretch in vitro. Using bioreactors containing cultured neurons, we recapitulated the peak biomechanical increase in embryonic rat crown-rump-length. Biologically paired sham and "stretch-grown" DRG neurons spanned 4.6- and 17.2-mm in length following static or stretch-induced growth conditions, respectively, which was associated with 456 significant changes in gene transcription identified by genome-wide cDNA microarrays. Eight significant genes found in DRG were cross-validated in stretch-grown cortical neurons by qRT-PCR, which included upregulation of Gpat3, Crem, Hmox1, Hpse, Mt1a, Nefm, Sprr1b, and downregulation of Nrep. The results herein establish a link between biomechanics and gene transcription in mammalian neurons, which elucidates the mechanism underlying long-term growth of axons, and provides a basis for new research in therapeutic axon regeneration.

Genomic signatures defining responsiveness to allopurinol and combination therapy for lung cancer identified by systems therapeutics analyses.

The ability to predict responsiveness to drugs in individual patients is limited. We hypothesized that integrating molecular information from databases would yield predictions that could be experimentally tested to develop transcriptomic signatures for specific drugs. We analyzed lung adenocarcinoma patient data from The Cancer Genome Atlas and identified a subset of patients in which xanthine dehydrogenase (XDH) expression correlated with decreased survival. We tested allopurinol, an FDA-approved drug that inhibits XDH, on human non-small-cell lung cancer (NSCLC) cell lines obtained from the Broad Institute Cancer Cell Line Encyclopedia and identified sensitive and resistant cell lines. We utilized the transcriptomic profiles of these cell lines to identify six-gene signatures for allopurinol-sensitive and allopurinol-resistant cell lines. Transcriptomic networks identified JAK2 as an additional target in allopurinol-resistant lines. Treatment of resistant cell lines with allopurinol and CEP-33779 (a JAK2 inhibitor) resulted in cell death. The effectiveness of allopurinol alone or allopurinol and CEP-33779 was verified in vivo using tumor formation in NCR-nude mice. We utilized the six-gene signatures to predict five additional allopurinol-sensitive NSCLC cell lines and four allopurinol-resistant cell lines susceptible to combination therapy. We searched the transcriptomic data from a library of patient-derived NSCLC tumors from the Jackson Laboratory to identify tumors that would be predicted to be sensitive to allopurinol or allopurinol + CEP-33779 treatment. Patient-derived tumors showed the predicted drug sensitivity in vivo. These data indicate that we can use integrated molecular information from cancer databases to predict drug responsiveness in individual patients and thus enable precision medicine.

Compartmentalized PDE4A5 Signaling Impairs Hippocampal Synaptic Plasticity and Long-Term Memory.

UNLABELLED: Alterations in cAMP signaling are thought to contribute to neurocognitive and neuropsychiatric disorders. Members of the cAMP-specific phosphodiesterase 4 (PDE4) family, which contains >25 different isoforms, play a key role in determining spatial cAMP degradation so as to orchestrate compartmentalized cAMP signaling in cells. Each isoform binds to a different set of protein complexes through its unique N-terminal domain, thereby leading to targeted degradation of cAMP in specific intracellular compartments. However, the functional role of specific compartmentalized PDE4 isoforms has not been examined in vivo Here, we show that increasing protein levels of the PDE4A5 isoform in mouse hippocampal excitatory neurons impairs a long-lasting form of hippocampal synaptic plasticity and attenuates hippocampus-dependent long-term memories without affecting anxiety. In contrast, viral expression of a truncated version of PDE4A5, which lacks the unique N-terminal targeting domain, does not affect long-term memory. Further, overexpression of the PDE4A1 isoform, which targets a different subset of signalosomes, leaves memory undisturbed. Fluorescence resonance energy transfer sensor-based cAMP measurements reveal that the full-length PDE4A5, in contrast to the truncated form, hampers forskolin-mediated increases in neuronal cAMP levels. Our study indicates that the unique N-terminal localization domain of PDE4A5 is essential for the targeting of specific cAMP-dependent signaling underlying synaptic plasticity and memory. The development of compounds to disrupt the compartmentalization of individual PDE4 isoforms by targeting their unique N-terminal domains may provide a fruitful approach to prevent cognitive deficits in neuropsychiatric and neurocognitive disorders that are associated with alterations in cAMP signaling. SIGNIFICANCE STATEMENT: Neurons exhibit localized signaling processes that enable biochemical cascades to be activated selectively in specific subcellular compartments. The phosphodiesterase 4 (PDE4) family coordinates the degradation of cAMP, leading to the local attenuation of cAMP-dependent signaling pathways. Sleep deprivation leads to increased hippocampal expression of the PDE4A5 isoform. Here, we explored whether PDE4A5 overexpression mimics behavioral and synaptic plasticity phenotypes associated with sleep deprivation. Viral expression of PDE4A5 in hippocampal neurons impairs long-term potentiation and attenuates the formation of hippocampus-dependent long-term memories. Our findings suggest that PDE4A5 is a molecular constraint on cognitive processes and may contribute to the development of novel therapeutic approaches to prevent cognitive deficits in neuropsychiatric and neurocognitive disorders that are associated with alterations in cAMP signaling.

Axon stretch growth: the mechanotransduction of neuronal growth.

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004). Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension. Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10 cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3 cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.