NEW IMAGING TECHNIQUE DISTIGUISHES TUMORS FROM NORMAL TISSUE

Even though the main objective of tumor surgery is to remove tumors as much as possible without disturbing the adjacent normal tissues, the task is very challenging in the operating room as neoplastic tissue is hard to distinguish from the adjacent healthy tissue. Thus, the portion of tumor still remained in the body after surgery causes recurrence, treatment failure, and poor outcome.

Surgery is an important treatment modality for brain tumors. Therefore, distinguishing normal tissue from tumor is extremely important for brain tumor surgery owing to the risk of damaging functional brain structures. Removal of healthy tissue can cause neurologic problems, but leaving tumor tissue behind can allow the cancer to grow and spread again. This is a major problem with glioblastoma multiforme (GBM), the most common form of malignant brain tumor in adults. Glioblastoma tumors grow quickly and are difficult to treat. The tumors infiltrate normal brain tissue and can’t be easily singled out. Therefore, tools designed to safely maximize the removal of tumor tissue are warranted. So far, experimental attempts to tell the difference between tumors and normal tissue during surgery have had limited success until recently, a new study by Ji et al. (2013) reported successful separation of  tumor-infiltrated brain tissue from surrounding healthy tissue in mice using stimulated Raman scattering (SRS) microscopy. Ji and colleagues provided evidence that SRS microscopy can be used to delineate tumor tissue in a human GBM xenograft mouse model, both ex vivo and in vivo, and in human brain tumor surgical specimens.

brain.scan_

Raman spectroscopy is a technique to study the interactions (vibrational, rotational, and other low-frequency modes) between matter and radiation in a system. It is named after the Indian noble laureate Dr. C.V. Raman (1930) who described the effect of light impinges upon a molecule and its interactions with the electron cloud and the bonds of that molecule. Chemical bonds in molecules have their own sets of vibration frequencies, and produce unique patterns of scattered light called Raman spectra. These spectra can be used as fingerprints to identify and differentiate different molecules in a complex environment. Developed on the concept of Raman spectroscopy, SRS is now emerging as an imaging technique to image biological tissues based on the intrinsic vibrational spectroscopy of their molecular components such as lipids, proteins, and DNA. Being free from the drawbacks of the dye-based methods, this label-free imaging technique exhibits high chemical selectivity enabling its use in complex biological applications including brain imaging.

To this end, Ji and colleagues used SRS microscopy to the problem of distinguishing protein-rich glioblastomas from more lipid-rich surrounding tissue and showed that it can be used to detect glioma ex vivo in human GBM xenograft mice, with results that correlated with the interpretation of hematoxylin and eosin (H&E)–stained slides by a surgical pathologist. Most importantly, this study demonstrated that SRS microscopy can detect extensive tumor infiltration in regions that appear grossly normal under standard bright-field conditions. This study suggests that SRS holds promise for improving the accuracy and effectiveness of cancer surgery. However, several challenges remain to be overcome including making a handheld surgical device with motion correction to acquire images from within a surgical cavity.

Reference:

Ji M, Orringer DA, Freudiger CW, Ramkissoon S, Liu X, Lau D, et al. Rapid, label-free detection of brain tumors with stimulated Raman scattering microscopy. Sci Transl Med. 2013;5:201ra119.

 

 

ISSCR 2013 Meeting Updates: Can Alzheimer’s Disease be modeled in a dish?

Human pluripotent stem cells (hPSCs) can differentiate into all cell types of the body, and can thereby serve as great models to examine the pathological mechanisms of various human diseases.  At the International Society for Stem Cell Research (ISSCR) 11th Annual Meeting, various stem cell experts highlighted the current human stem cell models for Alzheimer’s disease, and discussed the potential future directions of the field.

Alzheimer’s disease (AD) is the most common neurodegenerative dementia, affecting ~30 million people worldwide.  AD occurs in two main forms:  early-onset, familial AD (FAD) and late-onset, sporadic AD (SAD).  Both 40004_webare characterized by extensive neuronal loss and the aggregation of two proteins in the brain: amyloid β peptide (Aβ) and tau.  Aβ peptide is derived from the amyloid precursor protein (APP) via cleavage by two proteases, β-secretase and γ-secretase.  According to the amyloid cascade hypothesis, elevated levels of Aβ are necessary and sufficient to trigger disease 1.  Tau is synthesized in neurons and normally functions in binding to tubulin and stabilization of microtubules.  However, in AD, tau is hyper-phosphorylated, resulting in dissociation from microtubules, aggregation, and formation of neurofibrillary tangles (NFTs).  Although the pathological hallmarks of AD consist of these amyloid plaques and NFTs, how the two are related to each other and how they contribute to clinical onset and progression of AD is still under investigation.  By the time a patient manifests symptoms of a mild dementia, there is already significant neuronal loss and substantial accumulation of plaques and tangles.  One major limitation to our understanding of AD has been the lack of live, patient-specific neurons to examine disease progression.

With recent advances in reprogramming technology, scientists can now generate induced pluripotent stem cells (iPSCs), and thereby use live, patient-specific models to examine disease phenotypes in a dish.  At the ISSCR meeting, Larry Goldstein presented his lab’s recent work on using hiPSC models to study AD.  They generated iPSCs from two patients with FAD caused by a duplication of the APP gene, two patients with SAD, and two control individuals.  Next, neurons were generated from the iPSC lines by directed differentiation and fluorescence-activated cell sorting (FACS) purification 2.  Neurons from one SAD and two FAD patients demonstrated significantly higher levels of secreted Aβ and phosphorylated tau (p-tau) 3.  To determine whether there is an association between APP processing and elevated p-tau levels, they treated iPSC-derived neurons with γ-secretase and β-secretase inhibitors.  Interestingly, pharmacologic inhibition of β-secretase resulted in a significant reduction in the levels of Aβ and p-tau.  Treatment with the γ-secretase inhibitor only reduced Aβ levels, but not p-tau levels.  This suggests that products of APP processing other than Aβ might contribute to elevated p-tau levels, highlighting a potential weakness with the amyloid cascade hypothesis.

Other groups have proposed alternative hypotheses to explain AD pathogenesis.  Haruhisa Inoue presented his group’s work on using human iPSC models to examine how intracellular Aβ oligomers contribute to AD.  They generated iPSCs from one patient with FAD caused by the APP-E693Δ mutation, two patients with SAD, and three control individuals.  Corticol neurons were derived using small molecule inhibitors of bone morphogenic protein (BMP) and activin/nodal signaling as previously described 4.  Aβ oligomers accumulated in neurons derived from the FAD patient and one SAD patient, but not in the control neurons 5.  Specifically, the Aβ oligomers accumulated in the endoplasmic reticulum (ER), and triggered ER and oxidative stress in the neurons.  In addition, treatment with docosahexaenoic acid (DHA) alleviated the stress responses.  Although the drug has previously failed in some clinical trials of AD treatment, Inoue’s work suggests that DHA might be effective for a subset of patients.

In summary, Goldstein and Inoue presented convincing evidence that human iPSC models can be used to study early AD pathogenesis and patient-specific drug responses.  Although it can take decades for symptoms to manifest in patients, disease phenotypes can be observed using iPSC models.  However, the fact that only one out of two SAD patients generated a disease phenotype highlights the need of future iPSC studies to examine larger numbers of patients to account for the observed heterogeneity in AD pathogenesis.

References:

1          Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353-356, doi:10.1126/science.1072994 (2002).

2          Yuan, S. H. et al. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells. PLoS One 6, e17540, doi:10.1371/journal.pone.0017540 (2011).

3          Israel, M. A. et al. Probing sporadic and familial Alzheimer’s disease using induced pluripotent stem cells. Nature 482, 216-220, doi:10.1038/nature10821 (2012).

4          Morizane, A., Doi, D., Kikuchi, T., Nishimura, K. & Takahashi, J. Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells. J Neurosci Res 89, 117-126, doi:10.1002/jnr.22547 (2011).

5          Kondo, T. et al. Modeling Alzheimer’s disease with iPSCs reveals stress phenotypes associated with intracellular Abeta and differential drug responsiveness. Cell Stem Cell 12, 487-496, doi:10.1016/j.stem.2013.01.009 (2013).

 

 

Phase 1 Trial: Tolerance To MS Autoantigens Using Peptide-Coupled PBMCs

Multiple sclerosis (MS) is a degenerative inflammatory disease of the brain and the spinal cord, with its onset of symptoms occurring between the ages of 20 and 40. MS is categorized into two major forms: the most common form which accounts for 85%–90% of MS cases is relapsing-remitting MS (RRMS) whose victims usually develop secondary progressive MS (SPMS) over time. The second category, termed primary progressive MS (PPMS), accounts for approximately 10%–15% of MS cases that present with disability from the onset of the disease, progressing steadily with very little to no remissions in symptoms. It is not clear which factors are responsible for differentiating these different courses. In fact, up to date, there is little known about the underlying factors responsible for the complex heterogeneity, such as variation in immune abnormalities, observed among MS patients.Multiple sclerosis T cells

Although the etiology of MS remains unclear, it is predominantly considered to be driven by CD4+ T-cells autoreactivity to self-antigens expressed in the central nervous system (CNS), particularly to the myelin antigens. Three myelin sheath proteins that have been recognized as key autoantigens in MS include myelin basic protein (MBP), myelin oligodendrocyte protein (MOG), and proteolipid protein (PLP). Previous studies suggest epitope spreading may occur during the immune response to these three antigens in relapsing-remitting MS models. This notion is further supported by the existence of different target myelin epitopes in MS patients, which may be indicative of changes in the specificity of T-cell pathogenic response over time. These observations suggest the involvement of epitope spreading in MS, while providing a viable cause for the unfavorable efficacy reported from the several MS clinical trials that utilized a single antigen/peptidic-epitope in their therapeutic approach. In other words, previous clinical trials targeted pathogenic T-cells that are reactive against a single target antigen/epitope, which do not take into account the change of specificity in the pathogenic response overtime.

Antigen-coupled cell tolerance is a therapeutic approach aimed at antigen-specific T-cell tolerance via coupling target peptide(s) to carrier agents. In a recent study published in Science Translational Medicine, Lutterotti’s group report promising outcomes from their first-in-man MS clinical trial, demonstrating antigen-specific tolerance by autologous myelin peptide–coupled cells that utilizes a single infusion of autologous peripheral blood mononuclear cells (PBMCs) as the carrier cells. Seven myelin peptides which are believed to be key targets of autoreactive CD4+ T-cells in MS peptides (MOG1–20, MOG35–55, MBP13–32, MBP83–99, MBP111–129, MBP146–170, and PLP139–154), were chemically bound to the surface of patient-isolated PBMCs in the presence of the chemical cross-linker 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), followed by reinfusion back to the patient. Here, Lutterotti’s group validated the safety and feasibility of this antigen-coupled cell tolerization therapeutic approach in nine MS patients. Furthermore, they reported promising tolerability resulting from their approach, since patients’ immune autoreactivity to myelin peptides were reduced by 50 to 75 percent.  While all nine patients in this study displayed T-cell reactivity to at least one of the seven targeted myelin-peptides, seven were RRMS patients and two were SPMS patients.

 

describe the imageThese results support the epitope-spreading hypothesis, which indicates that MS patients make antibodies against one or a few myelin proteins, but as the disease progresses, the autoimmune response spreads to other myelin sheath epitopes. In their recent publication, Lutterotti et al. provide sufficient evidence necessitating emphasis not only on the specific target antigens, but also on the facility to inhibit epitope spreading, preferably prior to diversification of the CD4+ T-helper cell autoreactivity.

 

Lutterotti’s study is a significant step towards finding an effective strategy not only for treatment of MS, but also other T-cell-driven autoimmune disorders. Nonetheless, this therapeutic method must be tested on a much larger sample and geographically distinct population to demonstrate whether the efficacy reported here would be consistent in most if not all MS subtypes. The phase II of this clinical trial is planned to take place in the near future, during which more will be known about the long-term safety and efficacy of this technique. Regardless, the data presented by this study, at the very least, have set the requirement for future antigen-specific therapies to include the ability to target not only the previously activated autoreactive T-cells, but also the naïve autoreactive T-cells specific for several myelin epitopes.

Further Reading:

Antigen-Specific Tolerance by Autologous Myelin Peptide–Coupled Cells: A Phase 1 Trial in Multiple Sclerosis

MAPK Pathway Components: Modulators of Ataxin1 Toxicity in SCA1

amyloidWith the increasing prevalence of neurodegenerative disorders in the aging population, it has become more and more important to understand the molecular pathways that regulate and advance these disorders. Due to the high level of complexity of the mammalian brain, it is very difficult to devise improved targeted treatments. The biggest limitation in neurodegenerative disease research being the lack of viable biomarkers for the elder population. Neurodegenerative disorders such as Alzheimer’s, Parkinson’s and polyglutamine diseases, share many pathogenic abnormalities such as the accumulation of misfolded proteins due to mutations rendering them resistant to degradation or over-expression of the wild type form.

In the May 2013 issue of Nature, Dr. Zoghbi and colleagues at the Baylor College of Medicine, devised a strategy to identify therapeutic entry points that influence the levels of disease-driving proteins. They applied their approach to spinocerebellar ataxia type 1 (SCA1), a disease cased by expansion of the polyglutamine tract in ataxin 1 (ATXN1), using modulation of the ATXN1 pathway as a proof-of-principle. This model was chosen for several reasons: (1) neurodegeneration in SCA1 parallels with the levels of the mutant ATXN1 protein; (2) over-expression of wild type ATXN1 results in neurodegeneration; and (3) SCA1’s pathogenic mechanisms are well characterized. In order to identify regulators of ATXN1 levels the authors developed a human medullablastoma-derived cell line containing the transgene glutamine-expanded ATXN1 fused to red fluorescent protein (mRFP-ATXN1(82Q)).  Next, to distinguish modifiers that regulate ATXN1 protein levels from those affecting transgene transcription they included an internal ribosomal entry site followed by yellow fluorescent protein downstream of ATXN1 (mRFP-ATXN1(82Q)-IRES-YFP).  Their screen focused entirely on kinases and kinase like genes based on the fact that ATXN1 phosphorylation is known to be critical for its toxicity and because kinases are pharmacologically targetable. The authors tested 1908 small interfering RNAs (siRNAs) targeting 638 genes and assessing ATXN1 levels as a readout.  Subsequently, 50 siRNAs (corresponding to 45 genes) were selected based on their ability to reduce the ratio of RFP to YFP by 2 standard deviations from the mean.

A parallel genetic screen was performed using the Drosophila SCA1 model that expresses ATXN1(82Q). This model can be identified by an external eye phenotype. Here they screened 704 alleles (337 kinase encoding, including shRNA and loss of function mutations) for those that would modify ATXN1 levels. Based on morphological and histological assessments, they identified 51 alleles (49 genes) that suppressed ATXN1 toxicity in vivo. Additionally, human cell-based screens showed 10 human modifier genes that reduced ATXN1 and it’s associated toxicity, corresponding to 8 Drosophila modifiers.  Network analysis revealed that the MAPK cascade was the most enriched in both Drosophila and human, where 6/10 genes in human belonged to the canonical MAPK pathway (ERK1, ERK2, MED2, MEK3, MEK6, and MSK1).

ATXN1(82Q) is know to impair motor performance, thus, to determine the effects of the MAPK pathway on the central nervous system, a motor performance test was carried out in Drosophila. Decreasing the MEK, ERK1/2, and MSK1 Drosophila homologues by siRNA lead to increased motor performance and lifespan. Decreasing upstream MAPK pathway homologues suppressed ATXN1(82Q) eye defects and improved motor and lifespan phenotypes.  Conversely, constitutively active RAS exacerbated ATXN1 eye degeneration.  In human cells, decreasing HRAS and FNTA lead to decreased ATXN1 protein levels, and decreasing RAS homologues reduced ATXN1 in vivo.

Previous studies by Dr. Zoghbi’s group reported ATXN1 levels were sensitive to S776 phosphorylation.  Hence, they determined that of MAPK kinases implicated here, MSK1 would be able to phosphorylate the consensus sequence associated with S776.  To prove this, they performed an in vitro kinase assay with purified MSK1 and ATXN1 and found robust ATXN1-S776 phosphorylation in both mutant and WT protein forms. Next, cerebellar fractionation assays of WT mice revealed MSK1 was enriched and had increased activity in S776 phosphorylated fractions. Alternatively, immunodepletion of MSK1 from mouse cerebellar extracts lead to decreased S776 phosphorylation.

Next, they sought to determine whether the MAPK pathway could serve as a pharmacological target for SCA1. Human cells expressing ATXN1(82Q) were treated with a PDI84352 (MEK1/2 inhibitor), GW5704 (RAF1 inhibitor), and a Ro31-8220 (MSK1 inhibitor). Pharmacological inhibition of MAPK pathway lead to decreased ATXN1(82Q). Moreover, addition of MAPK inhibitors to cerebellar slices decreased ATXN1 levels.

Lastly, to test the genetic interaction between ATXN1 and MSK1, ATXN1(154Q) knock in mice (Atxn154Q/+) were bred to Msk1+/- Msk2+/- mice. Atxn154Q/+9 week old mice display a motor phenotype that can be quantified using a rotarod test.  Breeding of Atxn154Q/+ Msk1+/- Msk2+/- mice lead to better rotarod performance. Owing to the fact that ATXN1 alterations lead to Purkinje cell degeneration, they next determined whether eliminating one copy of MSK1 could rescue the loss of Purkinje cells in another mouse model of ATXN1(82Q), B05/+.  Indeed, single copy deletion of Msk1 lead to partially suppressed Purkinje loss phenotype and double MSK1 and MSK2 single copy deletion (B05/+Msk1+/- Msk2+/-), lead to decreased levels of ATXN1.

In summary, Dr. Zoghbi’s group have devised a proof-of-principle strategy that opens many new avenues for the identification of modifiers for neurodegenerative diseases. They utilized combined cross-species genetic screens to identify novel modifiers of ATXN1, and validated in human, mouse, and Drosophila models. This study focused on an early event in pathogenesis that could possibly delay disease onset and progression for this class of neurodegenerative disorders. The RAS-MAPK-MSK1 pathway’s role identified here (phosphorylation of S776-ATXN1) provides a novel pharmacological target for SCA1 and more importantly opens new avenues for combination therapies for this disease. Neurodegenerative disease research has primarily focused on developing treatments for advanced symptoms of neurodegeneration. It would be interesting to determine what the therapeutic benefits are of targeting the RAS-MAPK-MSK1 pathway are on a more advanced form of this disease and whether there would be at least partial reversion of motor defects.


References:

Park, J., et al., RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1. Nature.

Emamian,E.S.etal. Serine776 of ataxin-1is critical for polyglutamine-induced disease in SCA1 transgenic mice. Neuron 38, 375–387 (2003).

Jorgensen, N. D. et al. Phosphorylation of ATXN1 at Ser776 in the cerebellum. J. Neurochem. 110, 675–686 (2009).

Deep Brain Stimulation Shows Increased Cerebral Metabolism in Alzheimer’s Patients

Alzheimer’s disease (AD) is the most common form of dementia typically presenting itself after the age of 60, due to protein misfolding in the brain.  According to the National Institute on Aging, currently it is estimated that 5.1 million Americans suffer from AD. Dimentia, is defined as the loss of cognitive function including thinking, remembering, reasoning abilities, and behavioral abilities.  The severity of the disease ranges from pre-dementia (early onset) characterized by small affects on a persons functioning all the way to advanced where a person requires complete dependence on others for daily living.

AD was originally identified by Dr. Alois Alzheimer in 1906 after a brain biopsy of a woman showed abnormal clumps in her brain, now known as Amyloid plaques.  Amyloid plaques originate from amyloid-β (Aβ) deposits which are insoluble fibrous protein aggregates arising from misfolded proteins and polypeptides.

The dominant hypothesis for AD neuropathology is that Aβ plaque formations initiate a cascade that is followed by loss of neurons and synapses in the cerebral cortex and central subcortical regions as well as abnormal levels of neurotransmitters acetylcholine (decreased levels, leading to decreased cognition) and glutamate (increased levels, leading to neuronal over-activation and cell death).  The combined effects being progressive cell death in select regions of the brain. Currently available treatments for AD include drugs that target acetylcholine, glutamate and the amyloid cascade pathway.  These treatments are only efficacious at treating some of the symptoms they don’t stop the underlying decline and death of neurons. Additionally, it is now believed that Aβ plaques are necessary but not sufficient for the cognitive and neurodegenerative effects seen in AD patients. Therefore, the lack of efficacy in currently available treatments and the much needed understanding of the secondary effects due Aβ plaques, shows there is a dire need for safer and more efficacious therapies which can delay and reverse the effects of AD as well as modulate neuronal function in the affected neural circuits.

In a recent TEDx Talk, Dr. Andres M. Lozano from the Toronto Western Research Institute (TWRI) talks about a novel technology used for treating neuronal disorders, deep brain tissue stimulation (DBS). Dr. Lozano’s team have studied the effects of DBS on a variety of neurodegenerative disorders including AD and Parkinson’s disease showing exciting breakthroughs. DBS is surgical procedure where a brain pacemaker is implanted in an affected region of the brain.  The pacemaker can then be controlled externally and directed to send electrical impulses to a specific location.

The human brain consumes about 25% of the total body glucose levels.  In AD patients, it is well known that glucose uptake/metabolism is significantly impaired. Using positron emission tomography (PET) scans with the radiotracer [18F]-2-deoxy-2-fluoro-D-glucose (measures regional cerebral glucose metabolism) Dr. Lozano’s team found that DBS resulted in increased glucose metabolism in AD patients after 1 yr of treatment (http://clinicaltrials.gov/show/NCT00658125). More importantly, the increased metabolism correlated with better outcomes in global cognition, memory, and quality of life.  Based on preclinical studies, it is believed that DBS functions by inducing the generation of new neurons through electrical impulses.  Although, the effects of DBS on Aβ breakdown remain unclear and are under investigation in preclinical models.

It is important to note that not all patients displayed the same level of benefit from DBS.  In addition, all patients in this study had relatively mild AD and higher basal levels of cognitive function and glucose compared to more advanced patients. Therefore, it is possible that the effects of BDS in advanced AD patients will not be as beneficial, although this remains to be determined. It will be interesting to see what the long term effects of DBS will be and whether BDS will be sufficient to keep AD progression at bay.

Further Reading:

Increased Cerebral Metabolism After 1 Year of Deep Brain Stimulation in Alzheimer Disease. Gwenn S. Smith, Adrian W. Laxton, David F. Tang-Wai, Mary Pat McAndrews, Andreea Oliviana Diaconescu, Clifford I. Workman, Andres M. Lozano. Arch Neurol. 2012;69(9):1141-1148. doi:10.1001/archneurol.2012.590.

Memory rescue and enhanced neurogenesis following electrical stimulation of the anterior thalamus in rats treated with corticosterone. Clement Hamani, Scellig S. Stone, Ariel Garten, Andres M. Lozano, Gordon Winocur. Experimental Neurology.Nov;232(1):100-104. doi: 10.1016/j.expneurol.2011.08.023.

Potential of BDNF in Treating Neurodegenera­tive Disorders

Neurodegenerative diseases are characterized by usually fatal and progressive nervous system dysfunction caused by the death of neurons in the brain and spinal cord. In terms of human suffering and economic cost, neurodegenerative disorders carry an immense disease burden. However, despite extensive clinical research, especially in developing disease-modifying therapeutics, there is no effective medicine that halts or even slows any neurodegenerative disease. Currently in the United States, over 5 million Americans suffer from Alzheimer’s disease (AD), 1 million from Parkinson’s (PD), 400,000 from multiple sclerosis (MS), 30,000 from amyotrophic lateral sclerosis (ALS), and 30,000 from Huntington’s disease (HD). Thus, modification of current therapeutic research strategies and a more aggressive approach is a goal of increasing urgency.

clinical trials,alzheimer's,parkinson's,huntington's,clinical research,phase III

Thus far, the majority of clinical research for treatment of neurodegenera­tive diseases has utilized disease-modifying therapeutics, which either prevent or target elimination of the pathogenetic causes or neurotoxins resulting from the disease. The basis for this approach revolves around several characteristics implicated in neurodegenerative diseases, such as accumulation of neurotoxic substances, autophagy and inflamma­tion, as well as aggregation of misfolded proteins in neurodegenerative disorders, such as amyloid-β (Aβ) aggregates in AD or the mutant Huntington protein in HD, which take place prior to neuronal death. However, data obtained from several Phase III clinical trials indicate low efficacy of these treatments, specially in advanced stages of most neurodegenera­tive disorders; this is mainly due to the poor understanding of the underlying mechanisms of these disorders, hence the lack of knowledge of whether the targeted disease-characteristics are the cause or a symptom of the disease. Furthermore, the inability in early and accurate diagnosis of most neurodegenerative disorders impedes the early evaluation of therapeutic efficacy of new therapeutics.

Although the underlying cellular processes contributing to HD, PD and AD differ, one common denominator in all these neurodegenerative diseases is the presence of inadequate neuronal communication, induced by the loss of synapses. Neuronal communication is carried out via synaptic transmission at neuronal synapses. A change in the properties of synaptic transmission due to brain’s ability to dynamically reorganize itself by forming new neuronal synapses is referred to as synaptic plasticity; compensation for injury as well as adjustment of neural activity in response to new stimuli or changes in their environment are among the most critical known functions of synaptic plasticity. Thus, degeneration of synapses leads to the loss of synaptic plasticity, preventing neuronal stimulation and eventual cell death.

Alzheimer's Disease,Parkinson's,Huntington's,BDNF

According to a recent Review article published by scientists from GlaxoSmithKline in the advanced online edition of the Nature Reviews Neuroscience, Lu and colleagues present a compelling notion in treatment of neurodegenerative disorders; they propose a synaptic repair strategy targeting pathophysiology, which directly underlies the clinical syndromes. Unlike neuronal loss, synapse loss is reversible and synaptic dysfunction has the ability to be repaired, which allows the potential of neuronal repair prior to neuronal death. Furthermore, synaptic repair approach can be utilized for any neurological disease, regardless of the type or origin of the toxic byproduct. Lu’s group has proposed utilization of the synaptogenic molecule brain-derived neurotrophic factor (BDNF) as a potential synaptic repair therapeutic agent.

BDNF, an abundant neurotrophin expressed throughout the central nervous system, binds to NTRK2/TRKB and has been identified as one of the key neural signals regulating neuronal survival, neurogenesis and the only neurotrophin factor associated with synaptic plasticity in humans. Furthermore, in addition to its neuroprotective attribute, previous studies have demonstrated the key role of BDNF in cognitive functions, and synaptic deficit repair despite the presence of accumulated toxic proteins.

In this review, Lu’s team suggest potential routes of activating the BDNF pathway, as well as the importance of developing of a more reliable technique for quantification of synaptic changes in clinical settings, as essential tools in building effective disease-modifying therapies for neurodegenerative disorders. Although the notion of synaptic repair is an attractive one, the utilization of this strategy is still not feasible for the late stages of the disease, in which the irreversible neuronal death has already occurred. Furthermore, since most neurodegenerative diseases are diagnosed after the onset of neuronal death, emphasis on late stage treatment must remain a priority in neurodegenerative clinical research.

 

Further Reading:

BDNF-based synaptic repair as a disease-modifying strategy for neurodegenerative diseases

New in MS Research: Interplay Between IFN-beta, B-cells and Monocytes

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS), characterized by the presence of scar tissues (plaques) localized within the brain’s white matter and spinal cord. These plaques are results of myelin-degeneration (demyelination) and axonal death. Although MS has been classically considered to be a T-cell-mediated disease, the high efficacy of B cell-depleting therapies have demonstrated the critical role of B-lymphocytes and the humoral immune response in MS pathogenesis, albeit the underlying mechanisms remain unclear. In approximately 90% of MS patients, there is increased levels of intrathecally synthesized IgG in the MS-plaques as well as Cerebral Spinal Fluid (CSF), which manifests B-cell clonal expansions within the CNS.

B-cell-lineage cells differentiate into antibody-secreting plasma cells that are the source of persistent IgG, in the presence of key factors such as interleukin-6 (IL-6), B-cell-activating factor of the TNF family (BAFF) and a proliferation-inducing ligand (APRIL). IL-6 promotes terminal differentiation of B cells to plasma cells and is essential for the survival and Ig secretion. In conjunction with APRIL, BAFF regulates, B-cell survival, differentiation and is essential for initiation of T-cell independent B-cell responses. 

B cell resized 600

Type I IFNs (IFN-α, IFN-β, IFN-κ, and IFN-ω) are cytokines expressed by many cell types in response to viral or microbial infections, which bind to- and trigger specific Toll-like receptors (TLRs) that induce a large number of genes modulating and linking the innate and the adaptive immune responses.

Despite the development of other new treatments, IFN-β has been the first-line disease-modifying drug treatment for patients with relapsing-remitting multiple sclerosis (RRMS). Thus, understanding the molecular mechanisms of the anti-inflammatory effect of IFN-β in RRMS may provide insight into MS pathogenesis.  

TLRs are a family of non-catalytic pattern recognition receptors that recognize and bind to specific molecular patterns of pathogen-derived and endogenous damage-associated components. In addition to their key role in mediating innate immunity, TLRs have also been shown to play an important part in the activation of the adaptive immune system by inducing proinflammatory cytokines such as TNF-α, IL-1, IL-6, IL-12, and IFN.

Several studies have shown that of the 11 TLRs identified in humans, endosomal TLRs 7, 8 and 9 which recognize pathogen-derived and synthetic nucleic acids, also recognize endogenous immune complexes containing self-nucleic acids in certain autoimmune disorders such as MS. Interestingly, B-cells express both TLR7 and TLR9. TLR7 recognizes guanosine- and uridine-rich single-stranded RNAs (ssRNAs), whereas TLR9 recognizes hypomethylated CpG-rich double-stranded DNAs.  Upon activation by their specific ligands, these TLRs induce B cell proliferation and differentiation into Ig-secreting cells.

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In a recent study published in the European journal of Immunology, Coccia’s group has demonstrated the essential interactions between monocytes and B cells for the release of effective humoral immune response that elicits TLR7-mediated -induced B-cell differentiation into Ig secreting cells. Furthermore, they have shown a clear deficiency in this cross-talk interaction in MS patients; the peripheral blood mononuclear cell (PBMC) of MS patients exhibit substantially lowered TLR7-induced Ig production (compared to Healthy donors). However, results obtained after one-month long IFN-β therapy showed that lower humoral immune response in MS subjects was replenished, through IFN-β–induced secretion of TLR7- triggering cytokines, which mediated the selective increase in IgM and IgG to levels comparable to Healthy donors’. This data revealed that the IFN-β enhancement of TLR7-induced B-cell responses in MS patients occurs in at least two steps: 1) Regulation of TLR7 gene expression, and 2) Secretion of B-cell differentiation factors, in particular IL-6 and BAFF.

Finally, the last and perhaps the most significant finding of Coccia’s new study, is reporting, for the first time, the presence of a defect in TLR7 gene expression and signaling in monocytes of MS patients. Lack of TLR7-driven IgM and IgG production, absence of IL-6 and a significant reduction in BAFF expression in samples of MS patient-IFN-β treated PBMCs that were depleted of monocytes, evince IFN-β therapeutic mechanism by fine-tuning monocyte functions, through stimulation of TLR7 which subsequently effects B cell differentiation.

The discovery of the tight regulation of both TLR expression and TLR-induced responses in maintenance of immune environment’s homeostasis, as well as IFN-β-mediated- TLR7 function recovery are indicative of the critical changes in PBMC microenvironment induced by IFN-β therapy; within this microenvironment, leukocyte subsets establish critical immune regulatory interactions which determine the fate of the host’s immune tolerance processes.

Coccia’s new study has revealed new insights, which are not only crucial for the better understanding of the MS immunopathology, but also significant for development of new MS therapeutic strategies which target TLR expression and/or TLR-induced responses.


Further Reading:

IFN-β therapy modulates B-cell and monocyte crosstalk via TLR7 in multiple sclerosis patients.

Proof-of-principle study of the first-ever autologous iPSC-derived cell transplant in non-human primates

Shinya Yamanaka was awarded the Noble Prize for Medicine last year for his work on cellular reprogramming and creating induced pluripotent stem cells (iPSC).  Shinya Yamanaka found four transcription factors (Oct-3/4, Sox2, c-Myc, and Klf4) that determine pluripotency and he was able to reprogram differentiated adult cells into pluripotent cells that can then be re-differentiated into fully specialized tissue.  His findings raised great expectations, especially in the field of cellular therapy and regenerative medicine.  However, the path to the therapeutic use of iPSC is long and not without complications.  It was believed that iPSCs would avoid any immunogenic response because these cells can be developed from a patient’s own somatic cells. However, Zhao et al. challenged this notion when they discovered that iPSCs derived from C57BL/6 (B6) mice by the standard retroviral approach formed teratomas once transplanted into syngenic host mice and induced a rapid T cell dependent immune response1.

Recently, a group of researchers led by Dr. Su-Chun Zhang at the Waisman Center on the University of Wisconsin-Madison campus have shown that utilizing iPSCs as an autologous cell therapy is feasible and without any immunological reaction or rejection.  Dr. Zhang was the first to derive neural cells from embryonic stem cells (ESCs), as well as from iPSCs, and now has shown the first proof-of-principle that autologous iPSC-derived cells can engraft and survive in the primate brain.

In a new study published in Cell Reports, Zhang’s group reports on the successful generation of iPSCs from fibroblasts obtained from 8-10 year old rhesus monkeys (Macaca mulatta) using retroviruses containing the four Yamanaka factors, subsequent neuronal differentiation and cell transplantation back into the donor monkeys2.  The rhesus iPSCs were differentiated into neuroepithelia with the characteristic neural tube-like rosettes and expression of neuroectoderm transcription factors Pax6 and Sox1.  The neuronal rosettes were then expanded and further differentiated into neurons so that by the time for cell transplantation (day 42), 37% of the cells were bIII-tubulin+ neurons, 16% were S100b+ immature astrocytes, and 47% were Nestin+ progenitors.

primate ipsc cell pic
To examine the feasibility of transplanting autologous iPSC-derived neural progenitors in Parkinson’s disease, Zhang created parkinsonism in the rhesus monkeys by unilateral intracarotid artery injection of a neurotoxin 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP).  A year to 18 months after the MPTP infusion, all the monkeys developed a stable hemiparkinsonian condition distinguished by the characteristic tremors, bradykinesia, imbalance and impairment in motor skills.  At day 30 of cell culture the iPSC-derived neural progenitors were labeled with a GFP (green fluorescent protein) lentivirus and cells were transplanted at day 42 into the striatum and substantia nigra of the same monkey from which the iPSC were derived.

The monkey did not receive any immune suppression and engraftment of the GFP-labeled iPSC-derived neural progenitors was assessed at 6 months post-transplantation using stereological analysis on serial coronal sections.  Distinct grafts were present in the injected regions where 63% of the cells were microtubule-associated protein 2 (MAP2)+ neurons, 22% were glial fibrillary acidic protein (GFAP)+ astrocytes, and 10% were myelin basic protein (MBP)+ oligodendrocytes.  The GFP+ neurons showed long fibers extending into the surrounding host tissue and some neurons were present outside of the graft region expressing markers of mature neuronal differentiation.  Additionally, there was an absence of markers for pluripotent stem cells (OCT4, NANOG, SOX17, and Brachyury) and no positive staining for Ki67 (labels mitotic cells) indicating that the grafted progenitors had terminally differentiated.

Zhao et al. showed that the teratomas formed from transplanted iPSCs had an immunological rejection by syngenic mice1.  However, Zhang demonstrates that the autologous iPSC-derived neural cell transplants in the primate brain do not undergo immunological rejections suggested by the lack of CD3 and CD8 (lymphocyte markers) staining.  There was minimal response of endogenous glia (astrocytes and microglia) since staining for human leukocyte antigen D-related (HLA-DR; microglia and macrophage marker) was seen throughout the brain including the grafted regions.

Despite the positive engraftment and differentiation of the iPSC-derived neural progenitors, they did not see any behavioral improvement in the parkinsonian monkeys.  A potential explanation is that the GFP+ neurons were mostly g-Aminobutyric acid (GABA+) and few were positive for tyrosine hydroxylase (TH+).  TH catalyzes the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine.  Deficiency in TH has been implicated in giving rise to parkinsonian characteristics3.  Also, the number of transplanted cells may not have been enough to replace the dopamine-making cells in the primate brain.

However, this study provides hope for cell therapy using autologous iPSC-derived cells.  The iPSC-derived neural progenitors survived and differentiated into mature neurons, astrocytes, and oligodendrocytes in the primate brain with no evidence of immune rejection or teratoma formation.  The transplanted cells structurally integrated into the host brain and with characteristic features of neurons indicating by extending long processes and features of oligodendrocytes indicated by staining for myelin basic protein, suggestive of myelination.  This proof-of-principle study of the first-ever transplant of iPSC-derived cells back into the same non-human primate presents hope for personalized regenerative medicine and the neurodegenerative patient population.

Further reading:

1. Zhao, Tongbiao; Zhang, Zhen-Ning; Rong, Zhili; and Xu, Yang.  Immunogenicity of induced pluripotent stem cells.  Nature. 474, 212–215, 9 June 2011

2. Emborg ME, Liu Y, Xi J, Chang X, Yin Y, Lu J, Joers V, Swanson C, Holden JE, and Zhang Su.  Induced pluripotent stem cell-derived neural stem cells survive and mature in the nonhuman primate brain.  Cell Reports 3, 1-5, March 28, 2013.

3. Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens RC. Crystal Structure of tyrosine hydroxylase at 2.3a and its implications for inherited neurodegenerative diseases. Nature Structural Biology 4 (7): 578–585, 1997

Cell-based therapy for Parkinson’s disease: past, present and future.

Parkinson’s disease (PD) is a chronic neurodegenerative condition effecting dopaminergic neurons of the midbrain. PD manifests itself around age 50 with mainly motor symptoms, such as tremor (shaking), slowness of movement, rigidity and postural instability. Number of pharmaceutical agents (e.g., L-Dopa and MAO-B inhibitors) has been used for symptomatic relief in PD patients, but the ultimate therapy target is the replacement of degenerating dopaminergic neurons with new, healthy neurons.

describe the imageCell replacement therapy for PD dates back to mid 80s with the transplantation of adrenal medullary tissue into patients’ striatum [1-3], which resulted only moderate improvements. At the same time, researchers in Sweden performed transplantation of fetal ventral mesencephalic tissue from aborted fetuses [4, 5]. These early studies observed important and persistent improvement based on numerous clinical outcomes. Moreover, postmortem examination of the brains of PD patients, who received ventral mesencephalic tissue transplantation, showed sustained survival of the graft and re-innervation of the striatum [6]. With the lift of federal funding ban on using fetal tissue for research and therapy by President Clinton in 1993, United States also began clinical trials utilizing fetal ventral mesencephalic tissue [7, 8]. Unfortunately, not only the patients didn’t display any significant improvements following transplantation in these trials, they developed additional abnormal, involuntary movements (i.e., graft-induced dyskinesia), due to surgery, which was also observed in other trials.

Close examination of the transplantation studies using fetal ventral mesencephalic tissue revealed few noteworthy outcomes:

1. Younger patients with newly developed pathology showed significant improvements over older patients with severe PD pathology.

2. Some patients showed continued improvements 3-4 years after surgery, while they did not display any benefits during the first year, indicating that the improvement in clinical parameters may take a while to appear over time. Regardless, it is clear that patients respond differently to the transplants of dopaminergic neurons, making the clinical outcomes fluctuate considerably.

3. Preparation of the fetal tissues, as well as selection of patients for transplantation, varied significantly from center to center carrying out the clinical trials, further indicating the need for standardizing tissue preparation, patient selection and implantation site.

Compared to the aforementioned points, the use of fetal ventral mesencephalic tissue for grafting constitutes one of the biggest problems in cell based therapy for PD. It has been challenging to standardize the number and the quality of the fetal dopaminergic cells in graft preparations. Furthermore, the purity of the preparations also varies from batch to batch. Lastly, many ethical -and sometimes legal- issues surround fetal tissues/cells significantly limiting their clinical applicability. Do we have an alternative source that is free of these concerns/problems? The answer is yes, but not at the moment. With the isolation of human embryonic stem cells (hESCs) in 1998 and the introduction of human induced pluripotent stem cells (iPSCs) in 2007, stem cell derived dopaminergic neurons are at the top of everyone’s list when it comes to replacing degenerating neurons in PD. hESCs have been the primary source to produce dopaminergic neurons so far [9-11], but with the popularity and the advantages of iPSCs, the focus is more likely to shift to iPSC-derived dopaminergic neurons in future transplantation efforts.

Number of studies utilizing stem cell derived dopaminergic neurons in animal models of PD reported promising results over the years. However, we are far from using these cells in clinical trials. Many issues, such as long-term stability of the transplanted cells, sustained functional recovery, ability to re-innervate the host striatum, generation of GMP grade cells and long-terms safety especially with regards to tumor formation, remain to be determined. To be able to answer these concerns are critical for successful clinical translation of stem cell derived dopaminergic neurons. Nevertheless, the target is in front of everyone, and the field of regenerative medicine is moving at an incredible speed to reach it.  It should also be noted that an increasing number of novel therapeutic approaches (e.g., gene therapy and growth factor infusions) have been under development -in addition to cell transplantations- with the aim of restoring dopaminergic function in PD patients.

While we are looking ahead with the promise of stem cell derived dopaminergic neurons for future of cell-based therapy in PD, there are many lessons to be learnt from the early clinical trials using fetal ventral mesencephalic tissue. There is no question that fetal dopamine neurons will serve as a reference and a standard against stem cell derived neurons for future clinical trials, since we know that the transplants survived, re-innervated the striatum, and generated adequate symptomatic relief in some patients for more than a decade following surgery. For PD patients, who are interested in cell-based therapy now, the decision of whether to wait for clinical trials utilizing stem cell derived neurons or to proceed with currently available fetal tissue grafts remains a somewhat difficult question and should take into consideration the aforementioned strengths and weaknesses of each approach.

 

References:

[1] Backlund EO, Granberg PO, Hamberger B, et al. Transplantation of adrenal medullary tissue to striatum in parkinsonism. First clini- cal trials. J Neurosurg 1985;62:169–173.

[2] Herrera-Marschitz M, Stromberg I, Olsson D, Ungerstedt U, Olson L. Adrenal medullary implants in the dopamine-denervated rat striatum. II. Acute behavior as a function of graft amount and location and its modulation by neuroleptics. Brain Res 1984;297:53–61.

[3] Madrazo I, Drucker-Colin R, Diaz V, Martinez-Mata J, Torres C, Becerril JJ. Open microsurgical autograft of adrenal medulla to the right caudate nucleus in two patients with intractable Parkinson’s disease. N Engl J Med 1987;316:831–834.

[4] Lindvall O, Brundin P, Widner H, et al. Grafts of fetal dopamine neurons survive and improve motor function in Parkinson’s dis- ease. Science 1990;247:574–577.

[5] Widner H, Tetrud J, Rehncrona S, et al. Bilateral fetal mesence- phalic grafting in two patients with parkinsonism induced by 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). N Engl J Med 1992;327:1556–1563.

[6] Kordower JH, Rosenstein JM, Collier TJ, et al. Functional fetal nigral grafts in a patient with Parkinson’s disease: chemoanatomic, ultrastructural, and metabolic studies. J Comp Neurol 1996;370:203–230.

[7] Freed CR, Greene PE, Breeze RE, et al. Transplantation of embry- onic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001;344:710–719.

[8] Olanow CW, Goetz CG, Kordower JH, et al. A double-blind con- trolled trial of bilateral fetal nigral transplantation in Parkinson’s disease. Ann Neurol 2003;54:403–414.

[9] Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD. Effi- cient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–679.

[10] Cho MS, Lee YE, Kim JY, et al. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 2008;105:3392–3397.

[11] Kawasaki H, Suemori H, Mizuseki K, et al. Generation of dopami- nergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci U S A 2002;99:1580–1585.

Promising advances in Recurrent GBM treatment

Glioblastoma multiforme (GBM) are malignant brain tumors, classified by the World Health Organization (WHO), as grade IV tumors of neuroepithelial tissue and are the most common and deadly intracranial tumors, accounting for more than 70% of all brain tumors.  The current course of GBM treatment entails surgical resection followed by administration of radiation and chemotherapy.  However, despite this aggressive regimen and their devastating side effects on the patient, there are several obstacles that hinder their effectiveness; surgical resection of the primary tumor leads to injury to the surrounding normal tissue, while chemotherapy and radiotherapy cause toxicity to the healthy tissue in the brain. Furthermore, some anatomical feature unique to the central nervous system (CNS) such as the blood brain barrier (BBB) and the densely packed structure of brain’s parenchyma inhibits effective drug delivery throughout the brain.

GBM,glioma,FDA,brain tumors

Despite improvements in surgical techniques and post-operational delivery of chemotherapeutics and radiation, the prognosis for GBM patients remains dismal, making these lethal tumors virtually incurable. One of the most prominent intrinsic behaviors of GBM is its invasiveness, such that single glioma cells travel a distance from the tumor mass and invade adjacent brain tissue.  Due to the inherent capability for malignant glioma cells to rapidly proliferate and metastasize away from the primary tumor mass, surgical resection of the tumor is almost always followed by tumor recurrence (rGBM) with foci located as close as 1 centimeter from the resection cavity or as far as the opposite hemisphere. Although the history of glioma treatment dates back to the 19th century, the median survival of patients remains less than 14 months post-diagnosis.

Approximately two-third of rGBM patients are incapable of enduring additional surgical resections. Thus, for over 2 decades, Laser interstitial thermal therapy (LITT) has been used for extirpation of several malignant tumors, especially for patients with rGBM. LITT refers to utilization of low-powered thermal energy to locally cytoreduce the tumor tissue through transdermal thermocoagulation. Nonetheless, a number of technical barriers of the LITT device have impeded its widespread efficacy; two of these major barriers include lack of real-time monitoring of the tissue during treatment, as well as the control of the LITT energy (wavelength) output to correspond with the real time status of the brain tissue (healthy vs. tumor).

MRI,GBM,rGBM,brain tumors

In a recent study published in the Journal of Neurosurgery, Andrew Sloan’s group (Monteris® Medical) reported their results from the First-In-Man (FIM) Phase I clinical trial of their NeuroBlate™ Thermal Therapy System (also know as AutoLITT), which consists laser treatment of recurrent/progressive brain tumors in combination with intraoperative magnetic resonance imaging (MRI) technology. This system overcomes some of the major obstacles to LITT; without the use of radiation, it employs high-resolution MRI images of the brain in real time, which allows surgeons to visualize the progress of tumor ablation at all times, thus directing and controlling the laser deposition to increase treatment efficacy and minimizing harm to the surrounding healthy tissue.

The FDA has designated NeuroBlate™ Thermal Therapy System as safe, since no severe clinical toxicity or procedure-related neurological deficits were caused by this system. This Safety Trial was conducted on 10 patients (median age 55) with recurrent or progressive GBM in whom standard radiotherapy with or without chemotherapy had failed. Based on this study, not only NeuroBlate treatment did not cause any adverse side effects in the subjects, but it also resulted in higher response and survival times than expected.

The technological advancements of NeuroBlate™ Thermal Therapy System which allow a minimally-invasive, and possibly safer method of performing resections, not only for GBM, but perhaps also utilized for the future surgical resection of other cancers. Nonetheless, further investigation is needed to optimize NeuroBlate™ Thermal Therapy System in increasing the mean percentage of treated tumor at the intent-to-treat dose, and determine the long-term efficacy of this system compared with traditional surgical resection.

Further Reading:

Results of the NeuroBlate System first-in-humans Phase I clinical trial for recurrent glioblastoma