Lusine Danielyan

Intranasal delivery of cells to the brain

The safety and efficacy of cell-based therapies for neurodegenerative diseases depends on the mode of cell administration. We hypothesized that intranasally administered cells could bypass the blood-brain barrier by migrating from the nasal mucosa through the cribriform plate along the olfactory neural pathway into the brain and cerebrospinal fluid (CSF). This would minimize or eliminate the distribution of cellular grafts to peripheral organs and will help to dispense with neurosurgical cell implantation. Here we demonstrate transnasal delivery of cells to the brain following intranasal application of fluorescently labeled rat mesenchymal stem cells (MSC) or human glioma cells to naive mice and rats. After cells crossed the cribriform plate, two migration routes were identified: (1) migration into the olfactory bulb and to other parts of the brain; (2) entry into the CSF with movement along the surface of the cortex followed by entrance into the brain parenchyma. The delivery of cells was enhanced by hyaluronidase treatment applied intranasally 30 min prior to the application of cells. Intranasal delivery provides a new non-invasive method for cell delivery to the CNS.

Intranasal Treatment of Central Nervous System Dysfunction in Humans

ABSTRACTOne of the most challenging problems facing modern medicine is how to deliver a given drug to a specific target at the exclusion of other regions. For example, a variety of compounds have beneficial effects within the central nervous system (CNS), but unwanted side effects in the periphery. For such compounds, traditional oral or intravenous drug delivery fails to provide benefit without cost. However, intranasal delivery is emerging as a noninvasive option for delivering drugs to the CNS with minimal peripheral exposure. Additionally, this method facilitates the delivery of large and/or charged therapeutics, which fail to effectively cross the blood-brain barrier (BBB). Thus, for a variety of growth factors, hormones, neuropeptides and therapeutics including insulin, oxytocin, orexin, and even stem cells, intranasal delivery is emerging as an efficient method of administration, and represents a promising therapeutic strategy for the treatment of diseases with CNS involvement, such as obesity, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, depression, anxiety, autism spectrum disorders, seizures, drug addiction, eating disorders, and stroke.

Protective Effects of Intranasal Losartan in the APP/PS1 Transgenic Mouse Model of Alzheimer Disease

The local renin-angiotensin system (RAS) in the brain is a multitasking system controlling a plethora of essential functions such as neurogenic hypertension, baroreflexes, and sympathetic activity. Aside from its vasoactive actions, brain angiotensin II (AT-II) has also been implicated in the pathogenesis of cognitive decline, and beneficial effects of angiotensin receptor blockers (ARBs) in Alzheimer (AD) and Parkinson diseases (PD) are suggested. However, the use of ARBs at antihypertensive dosages would lead to unwanted hypotensive reactions in AD patients. Here we treated the APP/PS1 transgenic mouse model of AD with the ARB losartan (10 mg/kg body weight) to determine whether blockade of the AT-II receptor subtype 1 (AT1-R) with intranasal losartan, using at a dosage far below its systemic antihypertensive dose, could maintain its neuroprotective effects independent of its systemic vasoactive action. Intranasal losartan treatment (10 mg/kg every other day for 2 months) of APP/PS1 mice decreased amyloid beta (Abeta) plaques 3.7-fold. Blood serum levels of interleukin-12 (IL-12)p40/p70, IL-1beta, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in the vehicle-treated APP/PS1 mice. Intranasal losartan not only decreased IL-12p40/p70, IL-1beta, and GM-CSF, but also increased IL-10, which suppresses inflammation. Furthermore, losartan markedly increased tyrosine hydroxylase expression in the striatum and locus coeruleus. In conclusion, losartan exerts direct neuroprotective effects via its Abeta-reducing and antiinflammatory effects in the central nervous system (CNS). Therefore, intranasal losartan and potentially other ARBs, at concentrations below their threshold for altering systemic blood pressure, offer a new approach for the treatment of AD.

Intranasal Delivery of Bone Marrow-Derived Mesenchymal Stem Cells, Macrophages, and Microglia to the Brain in Mouse Models of Alzheimer's and Parkinson's Disease

In view of the rapid preclinical development of cell-based therapies for neurodegenerative disorders, traumatic brain injury, and tumors, the safe and efficient delivery and targeting of therapeutic cells to the central nervous system is critical for maintaining therapeutic efficacy and safety in the respective disease models. Our previous data demonstrated therapeutically efficacious and targeted delivery of mesenchymal stem cells (MSCs) to the brain in the rat 6-hydroxydopamine model of Parkinsons disease (PD). The present study examined delivery of bone marrow-derived MSCs, macrophages, and microglia to the brain in a transgenic model of PD [(Thy1)-h[A30P] aS] and an APP/PS1 model of Alzheimers disease (AD) via intranasal application (INA). INA of microglia in naive BL/6 mice led to targeted and effective delivery of cells to the brain. Quantitative PCR analysis of eGFP DNA showed that the brain contained the highest amount of eGFP-microglia (up to 2.1 × 104) after INA of 1 × 106 cells, while the total amount of cells detected in peripheral organs did not exceed 3.4 × 103. Seven days after INA, MSCs expressing eGFP were detected in the olfactory bulb (OB), cortex, amygdala, striatum, hippocampus, cerebellum, and brainstem of (Thy1)-h[A30P] aS transgenic mice, showing predominant distribution within the OB and brainstem. INA of eGFP-expressing macrophages in 13-month-old APP/PS1 mice led to delivery of cells to the OB, hippocampus, cortex, and cerebellum. Both MSCs and macrophages contained Iba-1-positive population of small microglia-like cells and Iba-1-negative large rounded cells showing either intracellular amyloid β (macrophages in APP/PS1 model) or α-synuclein [MSCs in (Thy1)-h[A30P] aS model] immunoreactivity. Here, we show, for the first time, intranasal delivery of cells to the brain of transgenic PD and AD mouse models. Additional work is needed to determine the optimal dosage (single treatment regimen or repeated administrations) to achieve functional improvement in these mouse models with intranasal microglia/macrophages and MSCs. This manuscript is published as part of the International Association of Neurorestoratology (IANR) special issue of Cell Transplantation.

Expression of blood group genes by mesenchymal stem cells

Incompatible blood group antigens are highly immunogenic and can cause graft rejections. Focusing on distinct carbohydrate‐ and protein‐based membrane structures, defined by blood group antigens, we investigated human bone marrow‐derived mesenchymal stem cells (MSCs) cultured in human serum. The presence of H (CD173), ABO, RhD, RhCE, RhAG, Kell, urea transporter type B (SLC14A1, previously known as JK), and Duffy antigen receptor of chemokines (DARC) was evaluated at the levels of genome, transcriptome and antigen. Fucosyltransferase‐1 (FUT1), RHCE, KEL, SLC14A1 (JK) and DARC mRNA were transcribed in MSCs. FUT1 mRNA transcription was lost during differentiation. The mRNA transcription of SLC14A1 (JK) decreased during chondrogenic differentiation, while that of DARC increased during adipogenic differentiation. All MSCs synthesized SLC14A1 (JK) but no DARC protein. However, none of the protein antigens tested occurred on the surface, indicating a lack of associated protein function in the membrane. As A and B antigens are neither expressed nor adsorbed, concerns of ABO compatibility with human serum supplements during culture are alleviated. The H antigen expression by GD2dim+ MSCs identified two distinct MSC subpopulations and enabled their isolation. We hypothesize that GD2dim+ H+ MSCs retain a better ‘stemness’. Because immunogenic blood group antigens are lacking, they cannot affect MSC engraftment in vivo, which is promising for clinical applications.

Age-Dependent Astroglial Vulnerability to Hypoxia and Glutamate: The Role for Erythropoietin

Extracellular accumulation of toxic concentrations of glutamate (Glu) is a hallmark of many neurodegenerative diseases, often accompanied by hypoxia and impaired metabolism of this neuromediator. To address the question whether the multifunctional neuroprotective action of erythropoietin (EPO) extends to the regulation of extracellular Glu-level and is age-related, young and culture-aged rat astroglial primary cells (APC) were simultaneously treated with 1mM Glu and/or human recombinant EPO under normoxic and hypoxic conditions (NC and HC). EPO increased the Glu uptake by astrocytes under both NC and especially upon HC in culture-aged APC (by 60%). Moreover, treatment with EPO up-regulated the activity of glutamine synthetase (GS), the expression of glutamate-aspartate transporter (GLAST) and the level of EPO mRNA. EPO alleviated the Glu- and hypoxia-induced LDH release from astrocytes. These protective EPO effects were concentration-dependent and they were strongly intensified with age in culture. More than a 4-fold increase in apoptosis and a 2-fold decrease in GS enzyme activity was observed in APC transfected with EPO receptor (EPOR)-siRNA. Our in vivo data show decreased expression of EPO and a strong increase of EPOR in brain homogenates of APP/PS1 mice and their wild type controls during aging. Comparison of APP/PS1 and age-matched WT control mice revealed a stronger expression of EPOR but a weaker one of EPO in the Alzheimer’s disease (AD) model mice. Here we show for the first time the direct correlation between the extent of differentiation (age) of astrocytes and the efficacy of EPO in balancing extracellular glutamate clearance and metabolism in an in-vitro model of hypoxia and Glu-induced astroglial injury. The clinical relevance of EPO and EPOR as markers of brain cells vulnerability during aging and neurodegeneration is evidenced by remarkable changes in their expression levels in a transgenic model of AD and their WT controls.

Keratinocytes as Depository of Ammonium-Inducible Glutamine Synthetase: Age- and Anatomy-Dependent Distribution in Human and Rat Skin

In inner organs, glutamine contributes to proliferation, detoxification and establishment of a mechanical barrier, i.e., functions essential for skin, as well. However, the age-dependent and regional peculiarities of distribution of glutamine synthetase (GS), an enzyme responsible for generation of glutamine, and factors regulating its enzymatic activity in mammalian skin remain undisclosed. To explore this, GS localization was investigated using immunohistochemistry and double-labeling of young and adult human and rat skin sections as well as skin cells in culture. In human and rat skin GS was almost completely co-localized with astrocyte-specific proteins (e.g. GFAP). While GS staining was pronounced in all layers of the epidermis of young human skin, staining was reduced and more differentiated among different layers with age. In stratum basale and in stratum spinosum GS was co-localized with the adherens junction component ß-catenin. Inhibition of, glycogen synthase kinase 3β in cultured keratinocytes and HaCaT cells, however, did not support a direct role of ß-catenin in regulation of GS. Enzymatic and reverse transcriptase polymerase chain reaction studies revealed an unusual mode of regulation of this enzyme in keratinocytes, i.e., GS activity, but not expression, was enhanced about 8–10 fold when the cells were exposed to ammonium ions. Prominent posttranscriptional up-regulation of GS activity in keratinocytes by ammonium ions in conjunction with widespread distribution of GS immunoreactivity throughout the epidermis allows considering the skin as a large reservoir of latent GS. Such a depository of glutamine-generating enzyme seems essential for continuous renewal of epidermal permeability barrier and during pathological processes accompanied by hyperammonemia.

Interplay between Endothelin and Erythropoietin in Astroglia: The Role in Protection against Hypoxia

We show that, under in vitro conditions, the vulnerability of astroglia to hypoxia is reflected by alterations in endothelin (ET)-1 release and capacity of erythropoietin (EPO) to regulate ET-1 levels. Exposure of cells to 24 h hypoxia did not induce changes in ET-1 release, while 48–72 h hypoxia resulted in increase of ET-1 release from astrocytes that could be abolished by EPO. The endothelin receptor type A (ETA) antagonist BQ123 increased extracellular levels of ET-1 in human fetal astroglial cell line (SV-FHAS). The survival and proliferation of rat primary astrocytes, neural precursors, and neurons upon hypoxic conditions were increased upon administration of BQ123. Hypoxic injury and aging affected the interaction between the EPO and ET systems. Under hypoxia EPO decreased ET-1 release from astrocytes, while ETA receptor blockade enhanced the expression of EPO mRNA and EPO receptor in culture-aged rat astroglia. The blockade of ETA receptor can increase the availability of ET-1 to the ETB receptor and can potentiate the neuroprotective effects of EPO. Thus, the new therapeutic use of combined administration of EPO and ETA receptor antagonists during hypoxia-associated neurodegenerative disorders of the central nervous system (CNS) can be suggested.

Neuroprotective, Neurogenic, and Amyloid Beta Reducing Effect of a Novel Alpha 2-Adrenoblocker, Mesedin, on Astroglia and Neuronal Progenitors upon Hypoxia and Glutamate Exposure

Locus coeruleus-noradrenergic system dysfunction is known to contribute to the progression of Alzheimer’s disease (AD). Besides a variety of reports showing the involvement of norepinephrine and its receptor systems in cognition, amyloid β (Aβ) metabolism, neuroinflammation, and neurogenesis, little is known about the contribution of the specific receptors to these actions. Here, we investigated the neurogenic and neuroprotective properties of a new α2 adrenoblocker, mesedin, in astroglial primary cultures (APC) from C57BL/6 and 3×Tg-AD mice. Our results demonstrate that mesedin rescues neuronal precursors and young neurons, and reduces the lactate dehydrogenase (LDH) release from astroglia under hypoxic and normoxic conditions. Mesedin also increased choline acetyltransferase, postsynaptic density marker 95 (PSD95), and Aβ-degrading enzyme neprilysin in the wild type APC, while in the 3×Tg-AD APC exposed to glutamate, it decreased the intracellular content of Aβ and enhanced the survival of synaptophysin-positive astroglia and neurons. These effects in APC can at least partially be attributed to the mesedin’s ability of increasing the expression of Interleukine(IL)-10, which is a potent anti-inflammatory, neuroprotective neurogenic, and Aβ metabolism enhancing factor. In summary, our data identify the neurogenic, neuroprotective, and anti-amyloidogenic action of mesedin in APC. Further in vivo studies are needed to estimate the therapeutic value of mesedin for AD.

Impaired Synthesis of Erythropoietin, Glutamine Synthetase and Metallothionein in the Skin of NOD/SCID/γcnull and Foxn1 nu/nu Mice with Misbalanced Production of MHC Class II Complex

Most skin pathologies are characterized by unbalanced synthesis of major histocompatability complex II (MHC-II) proteins. Healthy skin keratinocytes simultaneously produce large amounts of MHC-II and regeneration-supporting proteins, e.g. erythropoietin (EPO), EPO receptor (EPOR), glutamine synthetase (GS) and metallothionein (MT). To investigate the level of regeneration-supporting proteins in the skin during misbalanced production of MHC-II, skin sections from nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull and or Foxn1 nu/nu mice which are a priory known to under- and over-express MHC II, respectively, were used. Double immunofluorescence analysis of NOD/SCID/γcnull skin sections showed striking decrease in expression of MHC-II, EPO, GS and MT. In Foxn1 nu/nu mouse skin, GS was strongly expressed in epidermis and in hair follicles (HF), which lacked EPO. In nude mouse skin EPO and MHC-II were over-expressed in dermal fibroblasts and they were completely absent from cortex, channel, medulla and keratinocytes surrounding the HF, suggest a role for EPO in health and pathology of hair follicle. The level of expression of EPO and GS in both mutant mice was confirmed by results of Western blot analyses. Strong immunoresponsiveness of EPOR in the hair channels of NOD/SCID/γcnull mouse skin suggests increased requirements of skin cells for EPO and possible benefits of exogenous EPO application during disorders of immune system accompanied by loss MHC-II in skin cells.