Martin Marsala

Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells

Our understanding of Alzheimer’s disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer’s disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APPDp), two with sporadic Alzheimer’s disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APPDp patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1–40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APPDp and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer’s disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer’s disease, even though it can take decades for overt disease to manifest in patients.

Long-Distance Growth and Connectivity of Neural Stem Cells after Severe Spinal Cord Injury

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Activation of p38 mitogen‐activated protein kinase in spinal microglia is a critical link in inflammation‐induced spinal pain processing

We examined the effect of p38 mitogen‐activated protein kinase (MAPK) inhibitors in models of nociception and correlated this effect with localization and expression levels of p38 MAPK in spinal cord. There was a rapid increase in phosphorylated p38 MAPK in spinal cord following intrathecal administration of substance P or intradermal injection of formalin. Immuncytochemisty revealed that phosphorylated p38 MAPK‐immunoreactive cells were predominantly present in laminae I–IV of the dorsal horn. Double‐staining with markers for neurons, microglia, astrocytes and oligodendrocytes unexpectedly revealed co‐localization with microglia but not with neurons or other glia. Pretreatment with p38 MAPK inhibitors (SB20358 or SD‐282) had no effect on acute thermal thresholds. However, they attenuated hyperalgesia in several nociceptive models associated with spinal sensitization including direct spinal activation (intrathecal substance P) and peripheral tissue inflammation (intraplantar formalin or carrageenan). Spinal sensitization, manifested by enhanced expression of cyclo‐oxygenase‐2 and inflammation‐induced appearance of Fos‐positive neurons, was blocked by pretreatment, but not post‐treatment, with p38 MAPK inhibitors. Taken together, these results indicate that spinal p38 MAPK is involved in inflammation‐induced pain and that activated spinal microglia play a direct role in spinal nociceptive processing.

Down-regulation of the potassium-chloride cotransporter KCC2 contributes to spasticity after spinal cord injury

Hyperexcitability of spinal reflexes and reduced synaptic inhibition are commonly associated with spasticity after spinal cord injury (SCI). In adults, the activation of γ-aminobutyric acidA (GABAA) and glycine receptors inhibits neurons as a result of low intracellular chloride (Cl−) concentration, which is maintained by the potassium-chloride cotransporter KCC2 (encoded by Slc12a5). We show that KCC2 is downregulated after SCI in rats, particularly in motoneuron membranes, thereby depolarizing the Cl− equilibrium potential and reducing the strength of postsynaptic inhibition. Blocking KCC2 in intact rats reduces the rate-dependent depression (RDD) of the Hoffmann reflex, as is observed in spasticity. RDD is also decreased in KCC2-deficient mice and in intact rats after intrathecal brain-derived neurotrophic factor (BDNF) injection, which downregulates KCC2. The early decrease in KCC2 after SCI is prevented by sequestering BDNF at the time of SCI. Conversely, after SCI, BDNF upregulates KCC2 and restores RDD. Our results open new perspectives for the development of therapeutic strategies to alleviate spasticity.

Transcriptional signature and memory retention of human-induced pluripotent stem cells.

Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.

Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.

Background Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS). Methodology/Principal Findings We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184+/CD271−/CD44−/CD24+ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184−/CD44−/CD15LOW/CD24+ and a population of glia that was CD184+/CD44+ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo. Conclusions/Significance These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.

Effect of Proximal Arterial Perfusion Pressure on Function, Spinal Cord Blood Flow, and Histopathologic Changes After Increasing Intervals of Aortic Occlusion in the Rat

BACKGROUND AND PURPOSE Cross-clamping of the thoracic aorta results in spinal cord ischemia and prominent systemic hypertension. Using a rat model of transient spinal cord ischemia. we examined the effects of manipulation of proximal aortic blood pressure on spinal cord blood flow (SCBF), neurological dysfunction, and changes in spinal histopathology after increasing intervals of aortic occlusion. METHODS Aortic occlusion was induced by the inflation of a 2F Fogarty catheter placed into the thoracic aorta in rats anesthetized with halothane (1.5%). A tail artery was cannulated to monitor distal arterial pressure (DAP). To measure SCBF, a laser probe was implanted into the epidural space of the L-2 vertebra. To manipulate proximal arterial pressure (PAP), the left carotid artery was cannulated with a 20-gauge polytetrafluoroethylene catheter to permit blood withdrawal and infusion from a peripheral reservoir during aortic occlusion. In a survey study, spinal cord ischemia was induced in single animals at intervals of 6, 10, 15, 30, or 40 minutes with PAP controlled at 40, 60, 80, and 110 to 120 mm Hg. In a second series, ischemia was induced in groups of animals for 0, 6, 8, 10, and 12 minutes with PAP controlled at 40 mm Hg. After ischemia the animals survived for 2 to 3 days. During this recovery period, neurological functions were evaluated, followed by quantitative histopathology of the spinal cord. RESULTS Under normal conditions, cross-clamping yields an acute proximal hypertension (125 to 135 mm Hg), a fall of DAP to 15 to 22 mm Hg, and a decrease in SCBF to 7% to 11% of baseline values. With the use of the external reservoir, proximal hypertension could be abolished and the PAP maintained at target pressures. In these studies a typical syndrome of tactile allodynia, spastic paraplegia, and necrotic changes affecting the central part of the gray matter after 24 to 48 hours of reperfusion was observed at the following combinations of ischemic intervals and PAP values: > 10 minutes/40 mm Hg; > 12 minutes/60 mm Hg; > 16 minutes/80 mm Hg; and > 30 minutes/uncontrolled. Lowering PAP resulted in a corresponding decrease in residual SCBF. Systematic studies at a PAP of 40 mm Hg at occlusion intervals of 6, 8, 10, and 12 minutes revealed that 100% of rats were paraplegic after 10- and 12-minute ischemia, and these rats showed corresponding signs of spinal histopathology. CONCLUSIONS The present study shows that systemic intraischemic hypotension (40 mm Hg) significantly potentiates neurological dysfunction after transient aortic occlusion. The mechanism of the observed effect may include elimination of collateral flow during aortic occlusion and/or consequent potentiation of hypoperfusion during reperfusion. These data indicate that PAP during occlusion should be monitored and/or controlled because it is a critical variable in the determination of outcome in this model of spinal cord ischemia.

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Significance Mutations in the RNA binding protein TDP-43 cause amyotrophic lateral sclerosis and frontotemporal dementia. Through expressing disease-causing mutants in mice and genome-wide RNA splicing analyses, mutant TDP-43 is shown to retain normal or enhanced activity for facilitating splicing of some RNA targets, but “loss-of-function” for others. These splicing changes, as well as age-dependent, mutant-dependent lower motor neuron disease, occur without loss of nuclear TDP-43 or accumulation of insoluble aggregates of TDP-43. Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43Q331K and TDP-43M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.

The spinal loop dialysis catheter : characterization of use in the unanesthetized rat

To permit long-term measurement of time-dependent changes in levels of dialyzable drugs and transmitters in the spinal intrathecal (i.t.) space of the unanesthetized rat, we developed a dialysis catheter for chronic placement. This was accomplished by constructing a loop probe 9 cm in length from 0.3-mm-diameter dialysis tubing that was made impermeable except for the distal loop. This loop catheter was readily inserted though an incision in the cisternal membrane and passed to the lumbar enlargement. The ends of the catheter were then externalized on the top of the head. To permit i.t. injections, an additional i.t. catheter could also be inserted simultaneously by the same route. For dialysis, an external end of the loop catheter was connected to a syringe pump and perfused with artificial CSF (10 microliters/min) and the out flow collected. A series of studies were performed to demonstrate the characteristics and utility of this technique. (1) Stability of resting release: glutamate and glucose concentrations in spinal dialysate showed no significant changes from 3 to 10 days after implantation. (2) Spinal cord ischemia: ischemia induced by aortic occlusion or cardiac arrest evoked a time dependent increase in retrieved glutamate. (3) Spinal cord compression caused a time-dependent glutamate, aspartate and PGE2 increase. (4) Noxious afferent stimulation induced by the injection of formalin into the hindpaw resulted in a rapid and transient increase in dialysate glutamate concentration. (5) Direct activation of spinal excitatory amino acids receptors by i.t. injection of kainic acid (1 microgram) evoked a significant increase in aspartate and taurine. (6) Continuous delivery of spinal opiate (alfentanil) via dialysis resulted in a maintained, concentration dependent elevation in the thermal escape latencies in the unanesthetized rat. The loop dialysis catheter provides a robust experimental tool for studying time dependent changes in the concentration of diffusible substances in spinal CSF over an extended post-implantation interval and allows comparison of these changes with concurrently assessed behavioral indices.

Characterization of time course of spinal amino acids, citrulline and PGE2 release after carrageenan/kaolin-induced knee joint inflammation: a chronic microdialysis study

&NA; Pharmacological studies have implicated the spinal activation of excitatory amino acids, nitric oxide, and prostaglandins systems in the development of tactile and thermal hypersensitivity and central sensitization after peripheral inflammation. In the present study, using a chronically placed loop dialysis catheter, we examined in the unanesthetized rat the effect of carrageenan/kaolin (C/K)‐induced knee joint inflammation on the time course of spinal release of several active factors including excitatory amino acids (glutamate, aspartate), citrulline (a marker of nitric oxide formation), and prostaglandin E2 (PGE2) as well as the concomitant development of tactile and thermal hypersensitivity. Infection of C/K in the knee evoked a significant release of glutamate, with an initial peak seen immediately after knee C/K injection (179 ± 22%) and with a progressive and consistent increase over a period of 24 h (153–186%). Comparable changes in the concentration of aspartate (123–179%) were observed. Citrulline was constantly above baseline for the 24‐h period (121–158%). PGE2 was significantly increased at 10 min (146 ± 11%) with no change observed between 3–5 h. At 24 h, PGE2 was again significantly (143 ± 18%) increased. Behaviorally, a prominent thermal and tactile allodynia developed after injection with the peak seen by 1–3 h after induction of the inflammation. This hypersensitivity state, while diminished in its intensity, persisted for the entire observation period. These data suggest that increased spinal release of excitatory amino acids (EAA), nitric oxide and/or PGE2 is involved in the maintenance of the pain state initiated by acute peripheral inflammation.