2012:
2011:
- Inhibitors of TDP-43 Aggregation and Toxicity in ALS
- Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS
2010:
2009:
2012
Neuroprotective Small Molecules for the Treatment of Amyotrophic Lateral Sclerosis
Posted October 23, 2012
Serge Przedborski, Ph.D., Columbia UniversityAmyotrophic lateral sclerosis (ALS) is a debilitating neuromuscular disease for which there is no cure. The one substantive characteristic known about this disorder is that the sporadic form of the disease, which is also the more common form, is tied to mutations in the SOD1 gene. Thus far, more than 150 mutations have been documented in this gene, all of which lead to generally similar symptoms: muscle weakness, muscle wasting and eventual death. Research in Dr. Serge Przedborski's laboratory at Columbia University, and in other labs, has shown that it is the astrocyte that contains the mutated SOD1 that kills motor neurons (MNs) in the spinal cord. Further research in the Przedborski lab has shown that these mutated astrocytes produce a substance toxic to MNs which is also released into the surrounding culture medium that remains toxic even after the astrocytes are removed.
Dr. Przedborksi, recipient of an FY07 ALSRP Therapeutic Development Award, has developed a co-culture system containing MNs and conditioned medium from astrocytes to screen compounds for a therapeutically useful drug for ALS. Candidates that mitigated the toxic effects on MNs in the astrocyte-exposed medium would move forward for more extensive testing.
To validate the screening system, Dr. Przedborski's lab replicated their original findings that mutant SOD1 astrocyte-conditioned medium is toxic to primary spinal MNs and demonstrated that MNs exhibit selective vulnerability to the toxic media compared to other types of neurons. These effects were replicable with embryonic stem cell-derived motor neurons (ES-MNs), a preferable source, as they represent an endless supply of viable cells which make the model system readily expandable.
The initial targeted molecular screen has been fruitful, identifying JNK2/3 inhibitors as protective of MNs. A secondary low-throughput screen confirmed that JNK2/3 inhibitors are effective in protecting MNs. JNK inhibition has already been shown to reduce neuronal death and increase behavioral outcomes in neonatal brain injury. JNK seemed to have a role in apoptosis that could be the potential mode of cell death of the large MNs in ALS.
Bax inhibition via the peptide V5 was also found to protect MNs from the toxic astrocyte medium. When MNs from Bax, Bim, and Bak knockout mice were used with mutant astrocyte-conditioned medium, only Bax knockout primary MNs were unaffected by mutant SOD1 toxicity. Necrostatin-1 also offered protection to MNs, and the lab is now testing the potential benefit of necrostatin-1 use in an in vivo model of ALS, as well as trying to determine the effective levels of necrostatin in the blood, brain, and other tissues in these animals.
Dr. Przedborski's group continues to improve the statistical quality of the screening assay by increasing the signal-to-noise ratio from ~2 to almost 2.6, reflective of a high-quality assay with an excellent statistical power (0.80). The Z'-factor has also improved to 0.3, close to the recommended target of 0.50.Both the screen and the class of drug candidates it has already revealed could have a far-reaching impact towards the development of the first effective treatment for ALS.Links:
2011
Inhibitors of TDP-43 Aggregation and Toxicity in ALS
Posted November 10, 2011
Leonard Petrucelli, Ph.D., Mayo Clinic and Foundation, Jacksonville, Florida
Dr. Leonard Petrucelli's laboratory has pioneered neuroscience research aiming to understand the underlying mechanisms of amyotrophic lateral sclerosis (ALS) and identify potential drug targets for its treatment. TAR (Tat-responsive regulatory element) DNA-binding protein-43 (TDP-43) is a protein that has been found to go awry in approximately 90 percent of all ALS patients. Studies in yeast have revealed that C-terminal TDP-43 fragments are prone to aggregate and that only TDP-43 species that form inclusions, which result from continued protein aggregation, are toxic to neurons. With support from a Fiscal Year 2009 Therapeutic Development Award, Dr. Petrucelli is identifying compounds that prevent TDP-43 aggregation as potential neuroprotective agents for ALS. Using a previously developed human neuroblastoma cell line (M17D3) that overexpresses green fluorescent protein (GFP)-tagged C-terminal TDP-43 truncation product, GFP-TDP220-414, Dr. Petrucelli has begun screening compounds that reduce TDP220-414 aggregation, which is expressed as an attenuation of the GFP fluorescence. To increase the overall efficiency of the screening process, Dr. Petrucelli was able to effectively expand the assay from 24 to 384 wells. Over half of the 58,000 compounds from a select, proprietary small-molecule library have been screened on the M17D3 cells, and to date, 2,141 compounds have been found to attenuate GFP fluorescence (i.e., TDP fragment aggregation) by at least 30 percent. Expression of another relevant truncation product, GFP-TDP208-414, which can be exploited in M17D3 and also causes neurotoxicity in primary cortical neuronal cultures, is also being examined.
Future experiments will include further screening of the most promising compounds on primary cortical neuronal cultures. Expression of lactate dehydrogenase released into culture media will be measured as an indicator of cytotoxicity to further validate the compounds as potential therapeutic agents.Links:
Public and Technical Abstracts: Inhibitors of TDP-43 Aggregation and Toxicity
Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS
Posted April 7, 2011
Nicholas Maragakis, M.D., Johns Hopkins University, Baltimore, Maryland
Amyotrophic lateral sclerosis (ALS) is a degenerative disease affecting the upper and lower motor neurons in the brainstem and spinal cord. Neural degeneration from ALS leads to progressive loss of voluntary muscle function, then to paralysis, and ultimately to death. A recent development in stem cell technology called induced pluripotent stem cells, iPSCs, is helping scientists understand the abnormalities in the cell biology behind ALS. iPSCs start as skin cells harvested from an ALS patient that are re-programmed in culture (through exposure to certain transcription factors), first into stem cells that have the capacity to become any type of cell, and then differentiated into glial-restricted precursor cells (iPSC-GRPs). These iPSC-GRPs act like neural developmental stem cells and can become motor neurons, astrocytes, or oligodendrocytes in culture. These cells may ultimately be transplanted into patients to treat ALS. Evidence exists suggesting that astrocytes and other non-neuronal cell types play a role in the neurodegeneration of ALS. Replacement of astrocytes derived from iPSC-GRPs may offer a technically and biologically more feasible treatment modality for ALS patients compared with motor neuron transplantation.
Using funding from a Fiscal Year 2009 ALS Research Program Therapeutic Development Award, Dr. Nicholas Maragakis of Johns Hopkins is initiating pre-clinical studies of iPSC-GRPs to assess their therapeutic potential. Dr. Maragakis will examine whether human iPSC-GRPs derived from either sporadic ALS (sALS), familial SOD1-mediated ALS (fALS), or normal control subjects have the same capacity for engraftment, survival, and neuroprotective qualities following transplantation. It is not known whether iPSC-GRPs from ALS patients will be normal (and thus possibly neuroprotective) or whether these cells may harbor ALS-specific abnormalities which may lack benefit, or possibly even exacerbate disease. By comparing normal iPSC-GRPs with sALS iPSC-GRPs and fALS iPSC-GRPs, Dr. Maragakis will attempt to reveal inherent differences in astrocyte biology related to ALS, providing potential insight into ALS disease mechanisms. This initial assessment of the therapeutic potential of these cells will help determine whether continued investigation of this concept is warranted. Being able to use a patient's own cells to treat ALS (autologous cell transplantation) could preclude the need for significant immunosuppression, as well as decrease the probability of cell rejection.
In another phase of the study, Dr. Maragakis will build on previous studies in rats where mutant SOD1-GRPs transplanted into the cervical spinal cord of normal rats demonstrated initial feasibility for the proposed methodology. In vivo studies in this project will examine the activity of the different types of iPSC-GRPs (sALS, fALS, and normal) following transplantation into the spinal cords of normal rats. Survival, differentiation, migration, and other properties will be examined across the different cell types. These same cells will also be transplanted into the SOD1G93A rat model of ALS, where they will be compared for survivability and effects on motor neuron survival and muscular function.
Dr. Maragakis' study lays the framework to answer initial questions about properties of iPSCs from ALS patients through in vitro and in vivo comparative studies. It also offers an initial assessment of potential neuroprotection in an SOD1 animal model of ALS. These studies could represent the initial development of a viable autologous cell therapy for ALS patients.Links:
2010
Development of Lead Agents for ALS Treatment in Preclinical Model Systems Based on Differential Gene Expression of IGF-II
Posted February 26, 2010
Ole Isacson, M.D., McLean Hospital, Harvard Medical School
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that can be sporadic (the most common form ~ 90% of cases) and familial (hereditary form ~10% of cases). The neurodegeneration affects only somatic motor neurons (MNs) and not autonomic MNs. Studies in ALS mouse models have indicated that the initiation of neurodegeneration might be due to intrinsic factors associated with motor neurons, whereas astrocytes and microglia can also play an important role in the progression of neurodegeneration. Motor neuron subpopulations are prone to relatively differential vulnerability to neurodegeneration with similar pathology and pattern in both forms of ALS, whether sporadic or familial.
Dr. Ole Isacson has taken a novel approach to targeting ALS drug development by examining differential gene expression in subpopulations of motor neurons. He has previously applied this approach successfully in determining neuroprotection biomarkers in Parkinson's disease. His preliminary data from a rat model of ALS highlighted by cranial nerves oculomotor/trochlear (CN 3/4) complex, hypoglossal nerve (CN 12), and lateral motor column (LMC) MNs in symptomatic SOD1G93A rats versus wild-type rats indicated a slight decline of CN 12 MNs and a larger decline in LMC MNs in symptomatic SOD1G93A rats, while CN3/4 MNs seemed to be unaffected. Dr. Isacson then studied global gene and protein expression of CN3/4, CN12, and LMC of the cervical spinal cord in the normal rat. Analysis of in vitro functional assays demonstrated neuroprotective properties of insulin-like growth factor II (IGF-II) when used as a pretreatment for CN 3/4 MNs. IGF-II protected MNs from glutamate toxicity in a validated in vitro bioassay.
Building on these findings, Dr. Isacson, who received an ALSRP FY07 Therapeutic Development Award, has been developing a screening method for identifying compounds that can upregulate expression of IGF-II and that may have neuroprotective properties. High-throughput screening and polymerase chain reactions (PCR) are used to screen drug-like compounds from selected compound libraries (150,000 compounds) featuring many different drug categories. An initial screen of 1,040 generally FDA-approved drugs using quantitative PCR from MN cultures demonstrated 10% of these drugs have a twofold to sixfold upregulation of IGF-II. Notable drug candidates were found in anti-inflammatory, analgesic, and sex hormone-related drug categories. The high-hit compounds were further evaluated and selected by enhancement of IGF-II-related pathway phenotypes and by an in vitro glutamate toxicity assay, a validated bioassay for vulnerability to excitotoxic neuronal degeneration or MN death common in ALS.
Preliminary analysis of pharmacological and toxicological profiles of the selected candidate drugs are in progress both in vitro and in vivo. Additionally, selected candidate drugs with previously known brain permeability are being examined in both normal and pre-symptomatic SOD1G93A rats and mice for disease progression, behavioral and histopathological analysis. A larger screen of drug compounds with structural analysis and pharmacological and toxicological profiles in animal models will follow. The result of the large study will be an optimized candidate drug with high translational potential to be used as a first-line drug for ALS treatment.
Selected Recent Publications:
Chung CY, Koprich JB, Hallett PJ, and Isacson O. 2009. Functional enhancement and protection of dopaminergic terminals by RAB3B overexpression. Proc Natl Acad Sci U S A. 106(52):22474-22479. [Epub 2009 Dec 10.]
Isacson O. 2009. Cell therapy ahead for Parkinson's disease. Science 326(5956):1060.
Pruszak J, Just L, Isacson O, and Nikkhah G. 2009. Isolation and culture of ventral mesencephalic precursor cells and dopaminergic neurons from rodent brains. Curr Protoc Stem Cell Biol Chapter 2:Unit 2D.5.
Hedlund E and Isacson O. 2008. ALS model glia can mediate toxicity to motor neurons derived from human embryonic stem cells. Cell Stem Cell 3(6):575-576. Review.
2009
Neuroprotective Small Molecules for the Treatment of Amyotrophic Lateral Sclerosis (ALS)
Posted December 4, 2009
Serge Przedborski, Ph.D., Columbia University, New York
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive neurodegenerative disease characterized by the gradual degeneration and death of motor neurons in the brain and spinal cord. In the United States, 5,000 people are estimated to get diagnosed with ALS annually. Furthermore, U.S. military veterans reportedly have a higher risk of developing ALS. Approximately 90 to 95 percent of ALS cases are sporadic with no apparent risk factor, whereas 5 to 10 percent of ALS cases are hereditary and are known as familial ALS. Mutations in the gene encoding superoxide dismutase (SOD1), a potent antioxidant enzyme, are reportedly associated with about 20 percent of familial ALS cases. Dr. Serge Przedborski from Columbia University previously reported in vitro studies indicating that rodent astrocytes (non-neuronal cells surrounding neurons) expressing mutant SOD1 contribute to a more severe form of neurodegenerative process by killing spinal primary motor neurons and embryonic stem cell-derived motor neurons (ES-MNs). The death of motor neurons is accomplished through soluble neurotoxic factors mimicking the ALS phenotype compared to mutated SOD1 expression in primary motor neurons alone. Dr. Przedborski received a 2007 ALS Research Program Therapeutic Development Award to identify small molecule- neuroprotective agents for treatment of ALS. Dr. Przedborski along with collaborators Drs. Stockwell and Henderson at Columbia and Dr. Rubin at Harvard performed high-throughput screening of libraries of small compounds (about 30,000 small molecules at Columbia and 50,000 molecules at Harvard) to examine the individual effect at 10 µM concentration on the survival of mutant mouse ES-MNs cultured with rodent astrocytes expressing mutant SOD1. Promising compounds with high rates of ES-MNs survival were validated at both institutions, and were further tested at 10 different concentrations from 1 nM to 30 µM. After a comprehensive primary screening, 100 confirmed protective compounds were selected for secondary screening in the Przedborski laboratory using both rodent or mouse SOD1 mutant astrocytes and a mouse primary motor neuron model to study not only motor neuron survival rate but also neuronal protection of soma and processes by an assessment of axonal length. Translational potential of this on-going research is high. It should result in the identification of twenty most promising compounds with the highest potency, efficacy, and relevance to humans, which will move forward for pre-clinical and clinical studies that ultimately may result in a therapy for people living with ALS.
Selected Publications:
Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateral sclerosis mice
Banerjee R, Mosley RL, Reynolds AD, Dhar A, Jackson-Lewis V, Gordon PH, Przedborski S, Gendelman HE.
PLoS One. 2008 Jul 23;3(7):e2740.Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons
Nagai M, Re DB, Nagata T, Chalazonitis A, Jessell TM, Wichterle H, Przedborski S.
Nat Neurosci. 2007 May;10(5):615-22. Epub 2007 Apr 15.
