Advantages and Recent Developments of Autologous Cell Therapy for Parkinson's Disease Patients.

Parkinson's Disease (PD) is a progressive degenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. There are approximately 7-10 million PD patients worldwide. Currently, there are no biomarkers available or pharmaceuticals that can halt the dopaminergic neuron degeneration. At the time of diagnosis about 60% of the midbrain dopamine (mDA) neurons have already degenerated, resulting in a depletion of roughly 70% of striatal dopamine (DA) levels and synapses. Symptomatic treatment (e.g., with L-dopa) can initially restore DA levels and motor function, but with time often lead to side-effects like dyskinesia. Deep-brain-stimulation can alleviate these side-effects and some of the motor symptoms but requires repeat procedures and adds limitations for the patients. Restoration of dopaminergic synapses using neuronal cell replacement therapy has shown benefit in clinical studies using cells from fetal ventral midbrain. This approach, if done correctly, increases DA levels and restores synapses, allowing biofeedback regulation between the grafted cells and the host brain. Drawbacks are that it is not scalable for a large patient population and the patients require immunosuppression. Stem cells differentiated in vitro to mDA neurons or progenitors have shown promise in animal studies and is a scalable approach that allows for cryopreservation of transplantable cells and rigorous quality control prior to transplantation. However, all allogeneic grafts require immunosuppression. HLA-donor-matching, reduces, but does not completely eliminate, the need for immunosuppression, and is currently investigated in a clinical trial for PD in Japan. Since immune compatibility is very important in all areas of transplantation, these approaches may ultimately be of less benefit to the patients than an autologous approach. By using the patient's own somatic cells, reprogrammed to induced pluripotent stem cells (iPSCs) and differentiated to mDA neurons immunosuppression is not required, and may also present with several biological and functional advantages in the patients, as described in this article. The proof-of-principle of autologous iPSC mDA restoration of function has been shown in parkinsonian non-human primates (NHPs), and this can now be investigated in clinical trials in addition to the allogeneic and HLA-matched approaches. In this review, we focus on the autologous approach of cell therapy for PD.

Neurite Collapse and Altered ER Ca2+ Control in Human Parkinson Disease Patient iPSC-Derived Neurons with LRRK2 G2019S Mutation.

The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.

Increased motor neuron resilience by small molecule compounds that regulate IGF-II expression.

The selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is evident by sparing of a few subpopulations during this fast progressing and debilitating degenerative disease. By studying the gene expression profile of resilient vs. vulnerable motor neuron populations we can gain insight in what biomolecules and pathways may contribute to the resilience and vulnerability. Several genes have been found to be differentially expressed in the vulnerable motor neurons of the cervical spinal cord as compared to the spared motor neurons in CNIII/IV. One gene that is differentially expressed and present at higher levels in less vulnerable motor neurons is insulin-like growth factor II (IGF-II). The motor neuron protective effect of IGF-II has been demonstrated both in vitro and in SOD1 transgenic mice. Here, we have screened a library of small molecule compounds and identified inducers of IGF-II mRNA and protein expression. Several identified compounds significantly protected motor neurons from glutamate excitotoxicity in vitro. One of the compounds, vardenafil, resulted in a complete motor neuron protection, an effect that was reversed by blocking receptors of IGF-II. When administered to naïve rats vardenafil was present in the cerebrospinal fluid and increased IGF-II mRNA expression in the spinal cord. When administered to SOD1 transgenic mice, there was a significant delay in motor symptom onset and prolonged survival. Vardenafil also increased IGF-II mRNA and protein levels in motor neurons derived from healthy subject and ALS patient iPSCs, activated a human IGF-II promoter and improved survival of ALS-patient derived motor neurons in culture. Our findings suggest that modulation of genes differentially expressed in vulnerable and resilient motor neurons may be a useful therapeutic approach for motor neuron disease.

Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson's disease.

Autologous transplantation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons is a potential clinical approach for treatment of neurological disease. Preclinical demonstration of long-term efficacy, feasibility, and safety of iPSC-derived dopamine neurons in non-human primate models will be an important step in clinical development of cell therapy. Here, we analyzed cynomolgus monkey (CM) iPSC-derived midbrain dopamine neurons for up to 2 years following autologous transplantation in a Parkinson's disease (PD) model. In one animal, with the most successful protocol, we found that unilateral engraftment of CM-iPSCs could provide a gradual onset of functional motor improvement contralateral to the side of dopamine neuron transplantation, and increased motor activity, without a need for immunosuppression. Postmortem analyses demonstrated robust survival of midbrain-like dopaminergic neurons and extensive outgrowth into the transplanted putamen. Our proof of concept findings support further development of autologous iPSC-derived cell transplantation for treatment of PD.

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10µM), or large inclusions (≥10µM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.

Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations.

BACKGROUND: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. METHODOLOGY/PRINCIPAL FINDINGS: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D beta-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the binding constraints between the two proteins. Specific loss-of-function FLNa constructs were generated and used to analyze the importance of the FLNa-FilGAP interaction in vivo. Point mutagenesis revealed that disruption of the FLNa-FilGAP interface perturbs cell spreading. FilGAP does not bind FLNa homologs FLNb or FLNc establishing the importance of this interaction to the human FLNa mutations. Tight complex formation requires dimerization of both partners and the correct alignment of the binding surfaces, which is promoted by a flexible hinge domain between repeats 23 and 24 of FLNa. FLNa mutations associated with human developmental anomalies disrupt the binding interaction and weaken the elasticity of FLNa/F-actin network under high mechanical stress. CONCLUSIONS/SIGNIFICANCE: Mutational analysis informed by structure can generate reagents for probing specific cellular interactions of FLNa. Disease-related FLNa mutations have demonstrable effects on FLNa function.

Decreased levels of the gelsolin plasma isoform in patients with rheumatoid arthritis.

INTRODUCTION: Gelsolin is an intracellular actin-binding protein involved in cell shape changes, cell motility, and apoptosis. An extracellular gelsolin isoform, plasma gelsolin circulates in the blood of healthy individuals at a concentration of 200 +/- 50 mg/L and has been suggested to be a key component of an extracellular actin-scavenging system during tissue damage. Levels of plasma gelsolin decrease during acute injury and inflammation, and administration of recombinant plasma gelsolin to animals improves outcomes following sepsis or burn injuries. In the present study, we investigated plasma gelsolin in patients with rheumatoid arthritis. METHODS: Circulating and intra-articular levels of plasma gelsolin were measured in 78 patients with rheumatoid arthritis using a functional (pyrene-actin nucleation) assay and compared with 62 age- and gender-matched healthy controls. RESULTS: Circulating plasma gelsolin levels were significantly lower in patients with rheumatoid arthritis compared with healthy controls (141 +/- 32 versus 196 +/- 40 mg/L, P = 0.0002). The patients' intra-articular plasma gelsolin levels were significantly lower than in the paired plasma samples (94 +/- 24 versus 141 +/- 32 mg/L, P = 0.0001). Actin was detected in the synovial fluids of all but four of the patients, and immunoprecipitation experiments identified gelsolin-actin complexes. CONCLUSIONS: The plasma isoform of gelsolin is decreased in the plasma of patients with rheumatoid arthritis compared with healthy controls. The reduced plasma concentrations in combination with the presence of actin and gelsolin-actin complexes in synovial fluids suggest a local consumption of this potentially anti-inflammatory protein in the inflamed joint.

Structural basis of filamin A functions.

Filamin A (FLNa) can effect orthogonal branching of F-actin and bind many cellular constituents. FLNa dimeric subunits have N-terminal spectrin family F-actin binding domains (ABDs) and an elongated flexible segment of 24 immunoglobulin (Ig) repeats. We generated a library of FLNa fragments to examine their F-actin binding to define the structural properties of FLNa that enable its various functions. We find that Ig repeats 9-15 contain an F-actin-binding domain necessary for high avidity F-actin binding. Ig repeats 16-24, where most FLNa-binding partners interact, do not bind F-actin, and thus F-actin does not compete with Ig repeat 23 ligand, FilGAP. Ig repeats 16-24 have a compact structure that suggests their unfolding may accommodate pre-stress-mediated stiffening of F-actin networks, partner binding, mechanosensing, and mechanoprotection properties of FLNa. Our results also establish the orientation of FLNa dimers in F-actin branching. Dimerization, mediated by FLNa Ig repeat 24, accounts for rigid high-angle FLNa/F-actin branching resistant to bending by thermal forces, and high avidity F-actin binding and cross-linking.

Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin.

Gelsolin is a highly conserved intracellular actin-binding protein with an extracellular isoform, plasma gelsolin (pGSN). Blood concentrations of pGSN decrease in response to diverse tissue injuries. Depletion of pGSN to critical levels precedes and often predicts complications of injuries such as lung permeability changes and death. Administration of recombinant pGSN ameliorates such complications and reduces mortality in animal models. One proposed mechanism for pGSN's protective effects is that it inhibits inflammatory mediators generated during primary injuries, since pGSN binds bioactive mediators, including lysophospatidic acid (LPA) and endotoxin in vitro. However, no direct evidence in support of this hypothesis has been available. Here we show that recombinant pGSN modestly inhibited LPA-induced P-selectin upregulation by human platelets in the presence of albumin (P < 0.0001). However, physiologically relevant pGSN concentrations inhibit platelet-activating factor (PAF)-mediated P-selectin expression by up to 77% (P < 0.0001). pGSN also markedly inhibited PAF-induced superoxide anion (O(2)(-)) production of human peripheral neutrophils (PMN) in a concentration-dependent manner (P < 0.0001). A phospholipid-binding peptide derived from pGSN (QRLFQVKGRR) also inhibited PAF-mediated O(2)(-) generation (P = 0.024). Therefore, pGSN interferes with PAF- and LPA-induced cellular activation in vitro, suggesting a mechanism for the protective role of pGSN in vivo.