Category: Neurology

Several recent findings have identified variants in predominantly immune-response genes as underlying contributors in autoimmune diseases’ development. Nonetheless, along with other autoimmune disorders, there has been a significant increase particularly in incidences of multiple sclerosis (MS) and type-1 diabetes. These observations imply that in addition to the genetic causality, specific changes in environmental factors are significant contributors to the recent increases in autoimmune disorders.

Maintenance of immune system’s homeostasis is dependent on highly specified regulation of pro-inflammatory and regulatory CD4⁺ helper T-cell populations. T_H17 cells are pathogenic pro-inflammatory T-cells that produce a proinflammatory cytokine, Interleukin 17A (IL-17) and while they play a major role in immune defense against extracellular pathogens, they also play a major role in induction of autoimmune disorders, such as MS, psoriasis, rheumatoid arthritis, and type-1 diabetes, for which T-cell modulation is a common treatment regime.

Three previously identified factors, Interleukin 6 (IL-6), Transforming growth factor β (TGFβ) and Interleukin-23 subunit alpha (IL-23) are key players in the induction of naïve CD4⁺ T-cells to differentiate into T_H17 cells. However, the complete mechanism and factors responsible for stimulating naïve CD4⁺ T-cells’ differentiation into T_H17 cells is not well understood. Understanding of these underlying factors is crucial for developing therapeutic strategies to control T_H17 cell differentiation.

Three complementary, collaborative studies published in Nature this week report compelling insights on the regulatory mechanism of T_H17 cell differentiation and subsequent inflammatory response, while revealing evidence of interplay of genetics and a new environmental factor involved in autoimmune disease susceptibility.

By utilizing a unique approach, consisting of combination of transcriptional profiling, novel computational methods and nanowire-based short interfering RNA (siRNA) delivery to construct, Regev’s group was able to and identify 12 novel regulators and decipher the complex transcriptional network of T_H17 differentiation; they found that T_H17 differentiation is regulated by two intra-connected, but antagonistic networks, such that one module promotes T_H17 differentiation and proliferation while suppressing the development of other T-cells, whereas the antagonist module suppresses T_H17 cells.

By upregulating the expression of IL-23 receptor (IL-23R) on T_H17 cells, IL-23 sustains the T_H17 inflammatory response and induces its pathogenic effector functions. Through transcriptional profiling, Kuchroo’s group found that a serine/threonine kinase,  Serine/threonine-protein kinase 1 (SGK1), an essential node downstream of IL-23 signaling, is crucial for regulating IL-23R expression and stabilizing the T_H17 cell pathogenic effector function by inhibiting the molecule that represses IL-23R expression.

Previous studies have shown that SGK1 regulates Na⁺ transport and NaCl (salt) homeostasis in cells. Interestingly, Kuchroo’s study shows the positive correlation between increased concentration in salt and SGK1 expression, followed by upregulation of IL-23R and enhanced T_H17 differentiation in vitro as well as in vivo.

Consistent with Kuchroo’s finding, Hafler’s group reported that excess NaCl uptake can affect the innate immune system, and provided compelling data suggesting the possibility of the direct positive correlation between increased salt intake and incidence of autoimmune disease.

Previous studies have suggested the involvement of environmental factors, such as viruses, smoking, lack of sunlight and Vitamin D in different autoimmune disorders.  Salt, as the new potential environmental factor in autoimmune diseases is certainly an intriguing notion; not only due to the relevant molecular mechanisms reported in the three new studies, but also due to the fact that in most developed countries, the consumption of processed foods (such as fast foods), which contain over 100 times more NaCl compared to non-processed meals, has increased significantly over the past three decades. Nonetheless, future epidemiological studies in humans are needed to further investigate the possible correlation between salt consumption and autoimmunity incidence.

Further Readings:

Induction of Pathogenic TH17 Cells by Inducible Salt-sensing Kinase SGK1

Dynamic Regulatory Network Controlling TH17 Cell Differentiation

Sodium Chloride Drives Autoimmune Disease by The Induction Of Pathogenic TH17 Cells

Cancer is the formation of a malignant neoplasm, initiated by a cell that escaped apoptosis upon which mutations during DNA replication were not repaired; this cell, no longer regulated, continuously proliferates while each progeny will carry the previous mutation(s), while generating new ones, eventually resulting in a population of unregulated cells (malignant tumors) that will metastasize and take over the host’s body.  Thus, the conventional cancer treatment consists of surgical resection of the tumor mass, followed by administration of agents which kill any diving (mitotic) cells in the body (chemotherapy and radiation therapy). Consequently, these agents will not only target proliferating cancer cells, but they also destroy healthy proliferating cells, hence the side effects associated with chemotherapy, such as hair loss, nausea and cognitive deficits. This article emphasizes on chemotherapy’s role in disrupting processing speed, working memory and attention in humans.

Adult neurogenesis is the post-natal process of generating functional neurons (and glial cells) from adult neural precursors/progenitor cells (NPCs) throughout life. The two regions in the adult brain in which neurogenesis occurs are the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus. Newly generated cells in the SGZ can differentiate into functional neurons and integrate into the adult hippocampus’s DG as granule cells. Granule cells are involved in memory formation and many aspects of learning, with the exception of long-term memory storage.

Over 50% of cancer patients undergoing chemotherapy report significant cognitive impairment and declines in their overall cognitive processing, collectively referred to as “chemo-brain”. Thus, chemotherapy-induced loss of newly generated neurons in the hippocampus and impeding adult neurogenesis as the cause of such cognitive decline is a compelling notion. Furthermore, one of the highly “cognitive” oscillations in the human brain is the theta rhythm, which is mainly generated in the hippocampus and is also associated with processes of learning and memory. This rhythmic slow activity is also the most efficient synchronized electroencephalographic (EEG) activity that can be recorded from the brain.  It has been suggested that since synchronized oscillatory activity implements communication between functionally related structures during the process of learning, a chemotherapy-induced disruption in theta activity may obstruct inter-regional communication and result in learning deficits.

In a recent study published in the European Journal of Neuroscience, Shors’s group reported that prolonged systemic chemotherapy disrupts both the structural and functional integrity of the hippocampus, resulting in highly specific learning impairments. Their results show that chemotherapeutic agents instigate the learning deficits described in ‘chemo-brain’ via decreases in hippocampal adult neurogenesis and theta activity. Interestingly however, they are not responsible for the disruption of the hippocampus-independent memory for previously (pre-treatment) learned associations. In this study, the effects of chemotherapy on hippocampal adult neurogenesis, theta activity and learning were investigated through evaluating associative learning in adult male Sprague–Dawley rats by recording the hippocampal local-field potentials after several weeks of cyclic administration of the chemotherapeutic agent temozolomide (TMZ).The results revealed that TMZ’s effects on learning and theta activity were specific to a task in which an association had to be formed between temporally related but separate events, while no affects were observed in the expression of an already acquired trace memory.

TMZ is a small lipophilic monofunctional DNA alkylating agent, commonly used to treat metastatic malignant melanomas as well as tumors of the central nervous system (CNS), such as Glioblastoma Multiforme (GBM). Shors’s group also showed evidence of TMZ’s selective affect on neurogenesis, and not glia generation. They proposed the reason for this observation to be the possible differences in DNA repair mechanisms between neural precursors and glia. This notion is further supported by previous reports indicating that unlike TMZ, chemotherapeutic agents that do not readily cross the blood brain barrier (BBB), lower hippocampal neurogenesis and give rise to abnormal dendritic morphology. Additionally, it has been shown that cells surviving radiation therapy tend to differentiate into glial cells rather than neurons.

While most cancer patient undergoing chemotherapy experience short-term memory loss and difficulty performing complex tasks, about 15% of patients experience long-lasting cognitive problems due to long-term chemotherapy treatment. Identifying the underlying cause of some of these cognitive deficits is a major step towards finding of alternative agents or modifying current ones to eliminate or alleviate these issues.

Further Readings: 

Chemotherapy Disrupts Learning, Neurogenesis and Theta Activity in the Adult Brain.

Cognitive Side Effects of Cancer Therapy Demonstrate a Functional Role for Adult Neurogenesis.

Changing the Rate and Hippocampal Dependence of Trace Eyeblink Conditioning: Slow Learning Enhances Survival of New Neurons.

Multiple sclerosis (MS) is a chronically progressive, neuroinflammatory autoimmune disease of the central nervous system (CNS), mediated in part by CD4+ T-cells, which have escaped regulation and recognize myelin protein peptides.

CD4⁺ CD25⁺ regulatory T-cells (T_regs) are a subpopulation of suppressor T-cells that play a major role in maintenance of peripheral immune tolerance by active suppression of potential auto-aggressive T-cells. In contrast to patients with secondary progressive MS (SPMS) who have normal T_reg function, patients with relapsing-remitting MS (RRMS) have functionally impaired T_regs; this lack of regulatory suppression leads to the infiltration of pathogenic CD4 T-cells  into the CNS and the subsequent neuroinflammation.

Although patients with RRMS have a lower T_reg number and function, previous studies have shown no correlation between therapeutic response and increased T_reg number. However, based on data obtained from several autoimmune animal models, it has been speculated that the resistance of pathogenic CD4+ effector T cells (T_effs) to suppression by T_regs may be responsible for the failed tolerance in autoimmunity. Moreover, T_eff resistance has been reported in some human autoimmune diseases such as type 1 diabetes mellitus (T1D), rheumatoid arthritis (RA), and psoriasis. T_eff resistance is stimulated by several factors, including tumor necrosis factor–α (TNF-α), interleukin-4 (IL-4), IL-12, IL-6, IL-7, IL-15, IL-21 and the maturation state of CD4 T-cells.

In a recent study published in Nature, Schneider’s group demonstrated the presence of T_eff resistance in individuals with aggressive RRMS and the role of interleukin-6 (IL-6) in promoting T_eff resistance to T_regs.

Previous studies have shown the implication of IL-6 in MS pathology; IL-6 has been shown to inhibit apoptosis in T-cells, it is required for the differentiation of T-helper 17 (T_H17) cells, and local exposure to IL-6 can result in the development of T_effs resistant to suppression. During establishment of an immune response-derived inflammation, IL-6 levels elevate rapidly and bind to the IL-6 receptor α (IL-6Rα) on the CD4 T-cell’s surface. Next Glycoprotein 130 (gp130) is recruited to this IL-6-IL-6R complex, which results in activation and phosphorylation of the signal transducer and activator of transcription 3 (STAT3). Otherwise, IL-6 cytokine can bind soluble IL-6Rα (sIL-6Rα) in the serum and induce the phosphorylation of STAT3 by forming a complex that signals through membrane-bound gp130. In addition to the genetic correlation between variants in the STAT3 locus and MS susceptibility, a significant increase in phosphorylated STAT3 (pSTAT3) as well as IL-6Rα expression on CD4+ T cells, have been reported in RRMS patients.

In Schneider’s recent study, the role of T_eff resistance in RRMS’s failed tolerance was investigated by comparing T_effs from RRMS patients and healthy individuals, via T_reg suppression assays; the obtained results show that T_eff resistance is present only in the T_effs of RRMS patients with active disease (two or more clinical exacerbations or presence of one or more gadolinium-enhancing lesions on MRI within 2 years of sampling) and not those with inactive/mild disease.

Furthermore, by performing suppression assays in the presence of the STAT3 inhibitor (blocking STAT3 phosphorylation), they observed enhanced suppression, indicating a positive correlation between the degree of T_eff resistance and increased pSTAT3 in response to IL-6; their data imply that an increase in IL-6Rα expression on CD4+ T-cells and enhanced IL-6–mediated phosphorylation of STAT3 are major contributors to the impaired suppression observed among their RRMS subjects. They hypothesized that in active RRMS patients, the increased pSTAT3 and resistance of the pathogenic CD4 T-cells to regulation mediated by T_regs, is due to the elevated IL-6 production by microglia, astrocytes, endothelial cells, neurons, oligodendrocytes, or infiltrating T-cells in the CNS.

Schneider’s new findings suggest utilization of IL-6Rα expression and IL-6 mediated pSTAT3 as new therapeutic markers for determining disease activity as well as evaluating responsiveness to immunomodulatory therapies, such as tocilizumab (an IL-6Ra antagonist) in RRMS. Another significant aspects of this study is the unconventional technical approaches utilized in assessing the impact of IL-6 on suppression within an antigen-presenting cells (APCs)-free system, as well as ensuring consistency of activation and source of T_regs via a bead-based stimulation assay and in vitro–generated T_regs respectively. These unique techniques are key to the conclusions drawn from this study and useful for future MS research.

 

Further Reading:

In Active Relapsing-Remitting Multiple Sclerosis, Effector T Cell Resistance to Adaptive Tregs Involves IL-6-Mediated Signaling.   

In vitro Treg Suppression Assays.

Cell replacement therapy (CRT) and cell-based therapy (CBT) have provided promising therapeutic strategies for treatment of several human neurological diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD) and malignant gliomas (GBM).  The four most-studied cell types considered viable candidates for development of CRT and CBT for these neurological diseases consist of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs) and neural stem cells (NSCs). Although generation of different types of neurons and glial cells in vitro have been demonstrated by all these pluripotent cells, there are significant obstacles to the clinical utilization of stem cell-derived neurons or glial cells in CBT: First concern, aroused by previous studies, involves the long- term survival and phenotypic stability of stem cell-derived neurons or glial cells in vivo following transplantation. Second limitation is the high risk of any highly purified populations of neuronal cell type derived from ESCs, iPSCs, MSCs or NSCs, containing other neuronal/glial cell types, which may cause unfavorable interactions among grafted cells and/or with host central nervous system (CNS). Finally, the subpopulation (regardless of how small) of ESCs, iPSCs, MSCs or NSCs that did not completely differentiate, introduce a significant risk of tumorigenesis within the host CNS following transplantation. Furthermore, there are practical caveats, such as sustainable clinically approved, industrial quantity of these cells, which remain to be addressed.

In a recent review article published in the Journal of Neuropathology Seung U. Kim’s group have proposed utilization of immortalized human NSC lines as the cell-source for CBT in neurological diseases, as the best suited candidate. Kim’s group have previously generated clonally derived several immortalized human NSC lines, one of which has been particularly well characterized and currently used as a glioma therapy agent in phase II clinical trials. This particular line, named HB1.F3, was originally obtained from a fetal human telencephalon at 15 weeks gestation and immortalized by an amphotropic replication-incompetent retroviral vector, pLCN.v-myc, which encodes the v-myc oncogene.  This method of immortalization is not only safe, but also overcomes the issue of spontaneous differentiation, resulting in a non- tumorigenic, homogeneous NSC line.

HB1.F3’s exhibit normal human karyotype of 46XX, they are self-renewing and multipotent, capable of differentiating into neurons, astrocytes and oligodendrocytes, both in vivo and in vitro.  They express genes that encode for neurotrophic factors, such as for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF)-1, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), which can potentially make them a therapeutic agent rendering neuroprotection for neurons affected by injury or disease.

Kim’s group has reported functional improvement in a rat model of PD following HB1.F3 transplantation into the striatum. In yet another study, they show functional recovery in HD rat model, upon intravascular (iv) administration of HB1.F3s; their data suggests that the improvements observed here is due to the neuroprotection provided by HB1.F3s’ secretion of BDNF, since this factor has been previously shown to block neuronal injury under pathological conditions in animal models of HD. Another interesting outcome to this study is the integration of HB1.F3s in the striatum and homing to the site of neuronal injury, following their iv administration, indicative of their ability to freely cross the BBB. 

In AD patients, low levels of acetylcholine (ACh) is one contributing cause of cognitive impairment. The lack of sufficient ACh is due to the decreased activity of choline acetyltransferase (ChAT) that synthesizes ACh. Kim’s group transduced HB1.F3s, over-expressing the ChAT gene (F3.ChAT) and transplanted these NSCs into the brain of AD animal models. Their results show the functional recovery of presynaptic cholinergic system and fully restored learning and memory. Moreover, they generated motor neurons from HB1.F3s- encoding Olig2 basic helix loop helix (bHLH) transcription factor gene with sonic hedgehog (Shh) protein (F3.Olig2-Shh)- and transplanted them into L5 of the spinal cord of ALS animal model. This resulted in significantly delayed onset of the disease and prolonged average survival.

Utilization of HB1.F3s in human clinical trials was one of the first FDA permitted clinical trials in the United States, to use genetically modified human stem cells in maligant brain tumor CBT. Furthermore, the findings reported here do indicate that immortalized human NSCs are an effective source of cells for genetic manipulation and gene transfer into the CNS, for treatment of several neurological disorders. However, autologous iPSC-derived CNS cells seem to be a more promising strategy for CRT. This is mainly due to the risks associated with introducing immortalized cells, which may not survive long term post-transplantation. Nonetheless, all the mentioned stem cell sources have interesting characteristics that make each type suitable for treating different disorders.

 

Further Reading:

Neural Stem Cell-Based Treatment for Neurodegenerative Diseases

Contact and Encirclement of Glioma Cells in Vitro is an Intrinsic Behavior of a Clonal Human Neural Stem Cell Line.

Multiple sclerosis (MS) is believed to be a neurodegenerative autoimmune disorder, in which the body’s immune system attacks its own healthy tissue, specifically the myelin sheath surrounding the axons of the central nervous system (CNS). The word sclerosis refers to the scar tissues or the plaques within brain’s white matter, observed through the Magnetic Resonance Imaging (MRI) of MS patients’ brains. These plaques are results of myelin-degeneration (demyelination) and axonal death. The progression of MS symptoms is directly proportional to the failure of remylination by oligodentrocytes, leading to neurodegeneration; this demyelination disrupts the proper conduction of action potentials from CNS to different target organs and will eventually results in permanent disability caused by chronically demyelinated plaques. Some of the common symptoms include changes in sensation, muscle weakness, abnormal muscle spasms, or difficulty moving and maintaining balance, problems in speech or swallowing, as well as visual problems.

Therefore, in addition to inhibiting the autoimmunity, preventing permanent neurodegeneration as well as functional recovery of oligodentrocytes, are current therapeutic targets in MS clinical research. The focus of this article is to discuss the several recent studies that have reported promising results to this end.

Acid-Sensing Ion Channels (ASICs) are neuronal voltage-insensitive cationic channels activated by extracellular hydrogen ions (H+), and mediate entering and excessive accumulation of sodium (Na+) and calcium ions (Ca2+) inside the neuron’s cytoplasm. This intra-axonal accrual of Na+ and Ca2+ ions causes cellular injury and subsequent neurodegeneration in the CNS. Over-expression of ASIC1 has been observed in acute MS lesions (oligodendrocytes and axons with an injury co-express ASIC1 in chronic MS lesions) and believed to play a role in the development of irreversible tissue damage.

Moreover, amiloride, a potassium sparing diuretic (causes excretion of large amounts of potassium from the body), blocks ASICs by acting as “channel-blocker” and has been used for hypertension and congestive heart failure management. In a recent translational clinical study, effects of ASIC1-inhibition was tested in 14 patients with primary progressive MS, by comparing the rates of brain atrophy and tissue damage before and during amiloride treatment for 3 years. The results of this preliminary study show a significant decrease in the rate of whole-brain atrophy during the amiloride treatment period, which indicates reduced neurodegeneration (cell damage) through ASIC blocking. Although further studies with larger populations are needed to confirm the robustness of these observations, this is a safe, inexpensive promising potential neuroprotective MS treatment that may be utilized in conjunction with anti-inflammatory agents.

As mentioned previously, failure of oligodendrocytes to remyelinate leads to the severe clinical impairments associated with MS, which makes myelin- regeneration a significant therapeutic goal. Even though oligodendrocyte precursor cells are present, they fail to mature and myelinate in MS brain. Some of the key factors stimulating migration, maturation and survival of myelinating oligodendrocytes are components of extracellular matrix (ECM).

The ECM component in areas with MS lesions have significant differences when compared with the healthy adult brain tissue: two of these ECM abnormalities are the increased expression of Laminin, as well as upregulation of Fibronectin molecule that is absent in the normal brain’s white matter. Therefore, it has been speculated that fibronectin expression in the injury environment may inhibit oligodendrocyte maturation, contributing to remyelination failure in MS plaques.

A recent study suggests that the MS inflammation in the CNS causes astrocytes to accumulate fibronectin, which impair remyelination within the chronic lesions. These findings offer new strategic clinical approaches to promote remyelination through inhibiting fibronectin aggregation and its clearance from the inflammatory sites within the parenchyma.

Interestingly, another group have recently demonstrated a strong remyelinating effect of testosterone mediated by its receptor. They propose promotion of remyelination in males with MS, through utilizing synthetic drug which specifically bine the brain androgen receptors, employing testosterone as remyelinating agents.

Although there is no approved method to efficiently treat MS up to date, there are increasing reports on not only the disease’s underlying mechanisms, but also promising clinical strategies that are rapidly moving to human clinical trials. To learn about the role of the Blood Brain Barrier and other 2013 findings in MS, visit http://info.sanguinebio.com/neurology.

 

Klotho is a type I transmembrane protein, expressed in the brain, kidneys and reproductive organs; it is named after “Clotho”, a goddess from Greek mythology who “spins the thread of life” (the length of the threat is determinant of how long a certain individual will live). This denomination is due to the direct positive correlation between Klotho’s expression levels and life span/anti-aging phenotypes.

Klotho protein exists in two forms, membrane Klotho and secreted Klotho, and each form is associated with distinct functions. Some of Klotho’s age-suppressing functions include regulation of fibroblast growth factor-23 (FGF23) signaling, inhibition of insulin/insulin-like growth factor (IGF-1) and Wnt signaling pathways, and regulation of multiple cell-surface ion channels and growth factor receptors.

In contrast to other organ systems, the downstream effects of Klotho have not been as extensively studied within the central nervous system (CNS). This is surprising, considering that not only Klotho is present in serum and cerebrospinal fluid (CSF), but also is highly expressed in the hippocampus, choroid plexus and neurons. Disruption of the myelin sheath, either by activated pro-inflammatory cells or by protein defection within the oligodendrocytes has been previously described in aging brain, but the underlying factor stimulating the disruption was not clear. However, In 2008, Abraham’s group reported the significance of Klotho in age-related cognitive decline (ARCD), showing reduced expression of Klotho in regions of brain white matter as a function of age.

 

In January 2013, Abraham’s group reported their new findings regarding the effects of Klotho in oligodendrocyte maturation and CNS myelination, and its relation to ARCD. They showed Klotho‘s role in inducing oligodendrocytic progenitor cells (OPCs) maturation, by enhancing the expression of myelin proteins, such as myelin-associated glycoprotein (MAG), myelin basic protein (MBP), oligodendrocyte-specific protein (OSP/Claudin11), and proteolipid protein (PLP). Based on their in vivo studies, the loss of Klotho expression is correlated to defects in myelin that result in similar progressive axonal degeneration observed in hypomyelinating and demyelinating diseases, such as multiple sclerosis (MS).

Previous studies have shown Klotho’s role in facilitating removal of reactive oxygen species (ROS) and increasing resistance to oxidative stress. Furthermore, Nagai’s team observed impaired cognitive function in Klotho-deficient mice, as well as improved cognition upon treatment with the α-tocopherol antioxidant. Thus, Abraham’s group concluded that Klotho protein may function as a neuroprotective factor in the CNS through its antioxidative stress effect.

Together, these results provide strong evidence for Klotho protein as a key player, not only in myelin maintenance, but also in supporting oligodendrocyte and OPC function in the CNS; this makes Klotho an important member of the family of proteins responsible for neuron-oligodendrocyte communication. Abraham’s group hypothesized that downregulation of Klotho may be accountable for the observed white matter myelin degeneration-mediated ARCD, hence increasing Klotho protein expression can potentially prevent damage/protect myelin in the aging brain.

Abraham’s new findings are an exciting and important initial step towards development of new neuroprotective therapeutic strategies, such as induction of endogenous remyelination, for treatment of CNS diseases characterized by oligodendrocyte cell loss, such as MS and Schizophrenia. Additionally, early defects of insulin/IGF-1 receptor signaling in Alzheimer’s disease (AD), including the deficit of glucose metabolism that anticipates cognitive decline, may be partially due to deficiencies in Klotho levels. Further investigation on the precise mechanisms involved in Klotho’s regulation within the CNS seems promising for the future of neurodegenerative disease therapy.

 

Further Readings:

The Antiaging Protein Klotho Enhances Oligodendrocyte Maturation and Myelination of the CNS

Gene Profile Analysis Implicates Klotho As An Important Contributor To Aging Changes In Brain White Matter Of The Rhesus Monkey

The Putative Role of The Antiageing Protein Klotho in Cardiovascular and Renal Disease

Mesenchymal stem cells (MSCs) are multipotent cells present in, and can be isolated from a variety of adult tissues, such as bone marrow, umbilical cord blood or adipose tissue. MSCs have a number of advantageous characteristics that have made them a promising candidate for use in the new generation of cell-replacement therapy (CRT), even for central nervous system (CNS) disorders. Major obstacles to CRT in CNS disorders include successful delivery of therapeutic/stem cells to the damaged area/lesions within the CNS, host’s immune response against the allogenic cells and the possibility of ectopic tissue formation. MSCs exhibit unique characteristics which can overcome these obstacles, such as MSCs’ capacity to differentiate into multiple tissue-specific lineage cells, their role as progenitor-cell bioreactors of soluble factors that promote tissue regeneration from the damaged tissue and their immunomodulation capacity. Moreover, MSCs are considered immunoprivileged, meaning they have low expression of class II Major Histocompatibilty Complex (MHC-II) and other immune-stimulatory molecules on their cell surface. The focus of this discussion is on the utilization of MSCs in CNS-disease therapy, in particular multiple sclerosis (MS) and ischemic stroke (IS) .

Bone marrow derived (BM-MSCs) and adipose derived mesenchymal stem cells (AD-MSCs) have shown promising efficacy in an experimental autoimmune encephalomyelitis (EAE) preclinical model of MS, as well as the permanent middle cerebral artery occlusion (pMCAO) model of IS, respectively.

IS occurs as a result of an obstruction within a blood vessel supplying blood to a particular region of the brain, causing neuronal and astroglial damage within the immediate region. Replacement of these cells along with repairing the damaged tissue has been of great interest, turning IS clinical research towards stem cell therapy.

According to the recent study conducted by Gutierrez-Fernandez’s group, AD-MSCs are as restorative as BM-MSC in promoting recovery, repair and brain protection in IS rat models. They showed significant functional recovery, decreased apoptosis and increased expression of neurogenesis, synaptogenesis, angiogenesis and oligodendrogenesis markers, following the intravenous administration of allogenic AD-MSC and BM-MSC. Although these results suggest a less invasive route for administration of therapeutic cells, further studies addressing the fate of the administered cells are required, prior to clinical consideration of this method.

MS is a chronic, immune-mediated demyelinating disease of the CNS, characterized by demyelinated plaques within the brain and spinal cord. MS plaque formation consists of immune-cell infiltration, damage to oligodendrocytes and their subsequent failure to remyelinate, degeneration of axons, and ultimately astrocytosis. Up to date, there is no cure for MS; the current disease modifying therapies utilized to reduce the frequency/severity of relapses are limited to immunomodulatoion and are only partially effective. Thus, MS researchers have turned their attention to discovering potential therapeutics that will not only stop the autoimmune attacks, but also replace the destroyed CNS cells with properly functioning ones, through CRT.

Nonetheless, several recent studies have reported promising results regarding autologous, culture-expanded MSC transplantation in MS models. Based on Harris’s publication in Stem Cells Translational Medicine, intrathecal delivery of Bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) is a promising strategy for cell-based therapy in MS. They show that MSC-NPs derived from both, MS patients and healthy controls, uniformly displayed properties that support MSCs’ therapeutic potential in the CNS, regardless of the donor disease status. Like MSCs, MSC-NPs secrete immunomodulatory factors (such as cytokines and growth factors, including TGF-β, IL-6, IL-10, HGF, heme oxygenase-1, and nitric oxide) which inhibited T-cell proliferation and promoted naïve CD4⁺ T-cell polarization into FoxP3⁺ T cells. MSC-NPs also exhibit trophic effects similar to MSCs, by secreting trophic factors that promote oligodendroglial differentiation of neural stem cells (HGF, IGF-1, SDF1α, and VEGF). Furthermore, it was reported that MSC-NPs are neuroectodermally committed and have reduced capacity for mesodermal differentiation (reduced risk of abnormal tissue formation), which makes them a more suitable candidate for cell-based therapy in CNS injury.

One of the significant aspects of this study is the possibility of utilizing MS-patient’s own cells as the therapeutic cell source; not only because of the reduced immune response, but also due to the evidence of genetic stability of the adult stem cells present in these patients. This suggests several promising genetic and cellular therapeutic strategies to be investigated in the near future.

Further readings:

Characterization of autologous mesenchymal stem cell-derived neural progenitors as a feasible source of stemcells for central nervous system applications in multiple sclerosis.

Effects of intravenous administration of allogenic bone marrow- and adipose

tissue-derived mesenchymal stem cells on functional recovery and brain repair
markers in experimental ischemic stroke

Mesenchymal stem cell transplantation in multiple sclerosis.

Immunosuppressive properties of mesenchymal stem cells: advances and applications.

As mentioned in a previous article about Glioblastoma Multiforme (GBM), there are several obstacles that make these lethal brain tumors virtually incurable. Two hallmarks of malignant brain tumors are believed to be the major impediments to effective killing of all tumor cells via the current aggressive course of treatment: glioma cells’ exceptional invasivity and the heterogeneity of GBM, not only among individual patients, but also within a single tumor mass. Thus, these two glioma characteristics have generated great interest in GBM clinical research.

The malignancy of CNS tumors is categorized by the World Health Organization (WHO) grading system, ranging from grade I- grade IV based on the tumor’s “aggressiveness” (GBM is WHO grade IV astrocytoma).  However, unlike other neoplasms -in which local dissemination is usually limited and metastasis occurs via the vasculature or the lymphatic system– single glioma cells travel for several centimeters through adjacent brain tissue but will almost never establish systemic metastasis. Thus, the WHO tumor grade positively correlates with proliferation rate, rather than tumor invasion. This suggests the involvement of several independent genetic events that eventually lead to glioma progression, which is also consistent with the high levels of GBM heterogeneity. In order to develop a therapeutic agent that targets (finds and destroys) malignant glioma cells, it’s necessary to find a marker that’s expressed exclusively on the surface of these cells. Due to the high heterogeneity of GBM, as well as variations amnog individual patient brain tumors, no such marker has yet been identified.

There are compelling evidences of a subpopulation of malignant cells in GBM, which exhibit stem-cell like characteristics, such as multipotency, the ability to self-renew and invade; these tumor-initiating cells are referred to as cancer stem cells (CSC) and are believed to be responsible for tumor recurrence in GBM patients. Thus, researchers are no longer focusing on a single mutation/marker. Furthermore, it is becoming prominently important to rely on primary GBM patient samples for collecting data, since glioma cell lines do not represent the cellular and molecular components characteristic of the primary GBM.

CD133 (also known as Prominin-1 or AC133) is a penta-spanning membrane protein with two heavily glycosylated extracellular loops, and is recognized as a stem cell marker for certain normal and cancerous tissues. CD133 accumulates near the Golgi and ER and is also expressed on the cell surface. Although many previous studies reported that the presence of CD133+ glioma CSC subpopulation in GBM, drive tumor formation and its rapid proliferation, subsequent conflicting findings showed CD133-negative glioma cells’ ability to self-renew and form tumors in xeno-transplantation assays.

The recent findings by Brescia’s group confirm that cell-surface CD133 expression is a marker for self-renewing and tumor-initiating GBM cells, but a non-essential element for stem cell properties in all GBM cases. They show that even though membrane-bound CD133 were detectable only in a fraction of the patient samples (neurospheres and in freshly dissociated tumors), CD133 mRNA and intracellular CD133 protein were found expressed at high levels in almost all the examined neurosphere samples.

Through clonal analysis, they demonstrated the intrinsic capabilities of sinlge glioma cells and thier progeny: every clone derived from a single CD133-negative cell, contained a mixture of both CD133-negative and CD133-positive cells. Furthermore, they show an interesting interconvertible regulation of cell-surface CD133, through subcellular localization between the cytoplasm and the plasmamembrane. The localization of CD133 is likely determined by the tumor-associated microenvironmental cues.

The findings in this study have significant value in GBM research, taking one step closer to understanding the progression of malignant gliomas. However, the existence of the cytoplasmic CD133 reservoir and the re-cycling of this protein to the plasmamembrane and vise versa, hinders the therapeutic potential of using CD133 as a glioblastoma-targeting marker.

In a separate study published in Neuro-Oncology on the same week, researchers at Mayo Clinic introduced a significant association between malignant gliomas and Kallikrein 6 (KLK6) enzyme. KLK6 is a member of the kallikrein family of secreted serine proteases and has been reported to be elevated within areas of inflammation in CNS, suggesting its regulated expression with T-cell activation. Notably, the serum of Multiple Sclerosis (MS) patients contains elevated KLK6 levels as well. This study shows that as the KLK6 expression levels increase, the post-surgery survival times of GBM patients decrease. They found the highest levels of KLK6 were present in the most severe GBM. These results were obtained by looking at 60 samples of grade IV astrocytomas (thus categorized as GBM), and a less aggressive grade III astrocytomas.

 

Scarisbrick’s group also showed the possible role of KLK6 in promoting survival of malignant glioma cells, as well as increasing their resistance to apoptosis-inducing agents, such as radiotherapy (RT) and temozolomide (TMZ). The data supporting the pro-survival effect of KLK6 may introduce a new GBM therapeutic strategy, albeit the supporting experiments of these observations were conducted on U251 glioma cell line. If future studies report similar results that confirm the ability of KLK6 enzyme to promote primary patient tumor cells’ survival, then developing a therapeutic agent which targets KLK6’s function is a promising addition to GBM course of treatment ensuing the surgical resection of the tumor, preceding chemo-and radiotherapy.

Further Reading:

CD133 is essential for glioblastoma stem cell maintenance.

CD133 as a marker for regulation and potential for targete therapies in glioblastoma multiforme.

Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma.

Functional role of kallikrein 6 in regulating immune cell survival.

 

Multiple Sclerosis (MS) is a chronically progressive, neuroinflammatory autoimmune disease of the central nervous system (CNS), believed to be antigen-driven and predominantly T-cell-mediated. MS is characterized by demyelinated areas or patches of sclerosis (plaques) localized within the brain and spinal cord.

The normal physiological state of CNS is considered an anti-inflammatory environment, or “immune privileged”, which is partly due to passive-entry restriction of peripheral immune cells. During MS pathogenesis myelin-specific T-cells overcome these barriers, enter the CNS and recruit inflammatory cells that will eventually target and destroy the myelin protein, which leads to axonal damage.

Although the initial trigger leading to the development of myelin-specific T-cells in MS is still not clear, it has been shown that MS patients’ blood and cerebrospinal fluid (CSF), contain activated myelin‐reactive CD4+ T-cells, whereas only non-activated myelin‐reactive T-cells are present in non-MS samples. 

Both CD4+ and CD8+ T cells have been observed in MS acute and chronic lesions, respectively. Nonetheless, these two T-lymphocytes are activated by different CNS-resident antigen‐presenting cells (APCs) that trigger the recruitment of innate immune cells through presenting myelin antigens to CD4+ and CD8+ T cells, leading to the subsequent “determinant spreading”.

Previous studies have identified cells that present myelin to CD4+ T-cells: once inside the CNS, the CD4 molecule of autoreactive CD4+ T-cells binds to a non-polymorphic site on the major histocompatibility complex (MHC) class II, which is expressed by local myelin-presenting dendritic cells (DCs). In the absence of inflammation in CNS, there is a very low constitutive expression of MHC molecules, which are often present on cells of the lymphoid system.

In contrast CD8 molecule of the CD8+ T-cells binds to the MHC class I molecule, which serves to present specific antigens to the T-lymphocytes’s T-cell receptor (TCR). Thus, CD8+ T-cells (aka. cytotoxic T-cells) are involved in class I‐restricted lysis of antigen‐specific targets. However, until recently the APCs responsible for activating myelin-specific CD8+ T-cells were not known.

In January 2013, Goverman’s group from University of Washington showed that during experimental autoimmune encephalomyelitis (EAE)-an animal model of MS initiated by CD4+ T-cell- Tip-dendritic cells (Tip-DCs) play a major role in activating naive CD8+ T-cells. Based on this study, CD8+ T-cells are presented with MHC class I–restricted myelin basic protein (MBP) and activated by CD11b+ Tip-DCs. In addition, it was reported that under the inflammatory conditions of EAE, oligodendrocytes also presented MBP, to the CD8+ T-cells, which made them recognizable targets of the activated myelin-specific CD8+ T-cells, leading to sustained chronic inflammation (aka. Determinant spreading). This suggests that myelin-specific CD8+ T-cells may be responsible for the ongoing axonal destruction in “slow burning” MS lesions by directly lysing oligodendrocytes.

Tip-DCs are likely derived from the inflammatory monocytes that have accumulated in the brain and the spinal cord during EAE. Goverman proposed that one possible mechanism of acquiring MBP by Tip-DCs is via phagocytosing and processing myelin debris or dead oligodendrocytes, and then presenting the myelin peptides. Furthermore, they also hypothesized that CD8+ T-cells activated by Tip-DCs may contribute to the immune cascade amplification by secreting additional Interferon-gamma (IFN-γ) within CNS.

 

Identifying specific DCs involved in antigen-presenting and activation of all T-lymphocytes involved in the neuroinflammatory response is important for the development of potential autoimmune disease therapies that target immunogenic DC functions. Although Goverman’s findings may seem marginal on the surface, it is in fact a big step forward in understanding the etiology of MS; further investigations are required to address the origin of these Tip-DCs and the precise mechanisms through which they become myelin-presenting cells. Also, future studies in human MS are essential to confirm Tip-DCs’ reported functions as well as their interactions with patients’ oligodendrocytes.

Further reading:

MHC class I-restricted myelin epitopes are cross-presented by Tip-DCs that promote determinant spreading to CD8(+) T cells.

Antigen Presentation in the CNS by myeloid dendritic cells drives progression of relapsing experimental autoimmune encephalomyelitis. 

Dendritic cell CNS recruitment correlates with disease severity in EAE via CCL2 chemotaxis at the blood–brain barrier through paracellular transmigration and ERK activation.

 

Malignant gliomas are 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. One of the most prominent intrinsic behaviors of Glioblastoma Multiforme (GBM) is its invasiveness within the host’s central nervous system (CNS), in which single glioma cells travel a distance from the tumor mass and invade adjacent brain tissue

Due to the exceptional migratory ability of glioma cells, surgical resection of the tumor is almost always followed by tumor recurrence with foci located as close as 1 centimeter from the resection cavity or as far as the contralateral hemisphere. Despite advancements in surgical techniques and post-operational delivery of chemotherapeutics and radiation, the prognosis for glioma patients remains dismal making these lethal tumors virtually incurable. Although the history of glioma treatment dates back to the 19th century, the median survival of patients remains less than 14 months post-diagnosis. Thus, the only way to cure GBM is by essentially eliminating all glioma cells, including the single cells which have disseminated within the parenchyma, away from the tumor mass.

A promising potential strategy to treat high-grade brain tumors, is through cell-based therapies (CBTs) that incorporate autonomous tracking of tumor cells. Two examples of CBTs, which are currently under investigation in brain tumor clinical trials, include: 1) employing of genetically engineered neural stem cells (NSCs) as target-specific therapeutic-agent delivery vehicles, and 2) the adoptive transfer of tumor-specific, genetically engineered cytotoxic T-lymphocytes (CTLs).

 

The significance of NSCs and CTLs for treating CNS diseases: 

One anatomical feature unique to the CNS is the presence of the blood brain barrier (BBB), which restricts the access of many compounds including many chemotherapeutic agents into the CNS.  Thus, the BBB prevents effective drug delivery from the circulatory system to the tumor sites within the brain.  Even when the drug is administered intracranially to overcome the limitations presented by the BBB, the densely packed environment of brain’s parenchyma inhibits effective diffusion of the drug throughout the brain and prevents the drug from reaching the tumor cells. These anatomical properties of the CNS are also responsible for inefficient distribution of gene therapy in the brain.

NSCs readily cross the BBB and intrinsically target invasive tumor cells that have migrated away from the tumor mass in vivo. The HB1.F3 NSC line, developed by Dr. Karen Aboody, is one clonally derived human cell line that is particularly well characterized and is used clinically for glioma therapy. Another advantage of this therapeutic model is its efficacy through both intracranial and intravenous administration, without rejection elicited by the host’s immune system against the NSCs; this is due to HB1.F3 NSCs’ low levels of MHC Class I antigen expression and undetectable levels of MHC Class II antigens.

Previous studies have indicated CTLs’ ability to target and kill GBM, medulloblastomas and therapeutically resistant subpopulations of glioma stem–like cancer-initiating cells (GSC), which express interleukin-13 receptor α2 (IL13Rα2). Although the BBB is not permeable to CTLs, Dr. Christine E. Brown reported a non-toxic strategy of delivering these T-cells to the CNS tumors, by placing a fibrin matrix-embedded with CTLs, in the resection cavity during surgery.

In vivo,secretion of monocyte chemotactic protein-1 (MCP-1) also known as chemokine C-C motif ligand 2 (CCL2), by the cancer cells attract CD4+ and CD8+ T-cells, leading to the subsequent host antitumor immune response. Brown’s group has shown that the same tumor-secreted chemoattractants will recruit genetically engineered CTLs. Since CTLs migrate freely within fibrin matrices, the presence of MCP-1 in the surrounding environment attracts the CTLs to migrate out of the fibrin.  In their in vitro model, the IL13Rα2-specific T-cells successfully migrated out of the fibrin clot and killed the surrounding glioma cells.

Utilization of fibrin matrices allows a safe, non-toxic delivery of CTLs, without causing additional injury (i.e. injury caused by a catheter or needle injection) and inflammation to the brain tissue.

There are other therapeutic approaches for treatment of malignant brain tumors. Following are a list of further readings on the content of this article, as well as other current cancer research studies in regards to GBM:

 

 

 

Auffinger, B. et al. “New Therapeutic Approaches for Malignant Glioma: In Search of the Rosetta Stone.” F1000 Med Rep 4.18 (2012): doi: 10.3410/M4-18.

Brown, C.E., et al. “Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T cells.” Clinical Cancer Research, 18 (8), (2012) pp. 2199-2209

Khosh, N., et al. “Contact and Encirclement of Glioma Cells in vitro is an Intrinsic Behavior of a clonal Human Neural Stem Cell Line.” PLoS ONE, 7 (12) (2012) . doi:10.1371/ journal.pone. 0051859

Zou, Z., et al. “Cytotoxic T Lymphocyte Trafficking and Survival in an Augmented Fibrin Matrix Carrier.” PLoS ONE, 7(4) (2012). doi:10.1371/journal.pone.0034652

Aboody, K., et al. “Translating stem cell studies to the clinic for CNS repair: current state of the art and the need for a Rosetta stone.” Neuron, 26 May 2011 (Vol. 70, Issue 4, pp. 597-613)