Silvia Bolognin

Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells

Summary Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson’s disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

Rescue of cognitive-aging by administration of a neurogenic and/or neurotrophic compound

Aging is characterized by a progressive decline of cognitive performance, which has been partially attributed to structural and functional alterations of hippocampus. Importantly, aging is the major risk factor for the development of neurodegenerative diseases, especially Alzheimers disease. An important therapeutic approach to counteract the age-associated memory dysfunctions is to maintain an appropriate microenvironment for successful neurogenesis and synaptic plasticity. In this study, we show that chronic oral administration of peptide 021 (P021), a small peptidergic neurotrophic compound derived from the ciliary neurotrophic factor, significantly reduced the age-dependent decline in learning and memory in 22 to 24-month-old Fisher rats. Treatment with P021 inhibited the deficit in neurogenesis in the aged rats and increased the expression of brain derived neurotrophic factor. Furthermore, P021 restored synaptic deficits both in the cortex and the hippocampus. In vivo magnetic resonance spectroscopy revealed age-dependent alterations in hippocampal content of several metabolites. Remarkably, P021 was effective in significantly reducing myoinositol (INS) concentration, which was increased in aged compared with young rats. These findings suggest that stimulating endogenous neuroprotective mechanisms is a potential therapeutic approach to cognitive aging, Alzheimers disease, and associated neurodegenerative disorders and P021 is a promising compound for this purpose.

Rapid and robust generation of long-term self-renewing human neural stem cells with the ability to generate mature astroglia

Induced pluripotent stem cell bear the potential to differentiate into any desired cell type and hold large promise for disease-in-a-dish cell-modeling approaches. With the latest advances in the field of reprogramming technology, the generation of patient-specific cells has become a standard technology. However, directed and homogenous differentiation of human pluripotent stem cells into desired specific cell types remains an experimental challenge. Here, we report the development of a novel hiPSCs-based protocol enabling the generation of expandable homogenous human neural stem cells (hNSCs) that can be maintained under self-renewing conditions over high passage numbers. Our newly generated hNSCs retained differentiation potential as evidenced by the reliable generation of mature astrocytes that display typical properties as glutamate up-take and expression of aquaporin-4. The hNSC-derived astrocytes showed high activity of pyruvate carboxylase as assessed by stable isotope assisted metabolic profiling. Moreover, using a cell transplantation approach, we showed that grafted hNSCs were not only able to survive but also to differentiate into astroglial in vivo. Engraftments of pluripotent stem cells derived from somatic cells carry an inherent tumor formation potential. Our results demonstrate that hNSCs with self-renewing and differentiation potential may provide a safer alternative strategy, with promising applications especially for neurodegenerative disorders.

Interaction between Alzheimer's Amyloid-β and Amyloid-β-Metal Complexes with Cell Membranes

A number of observations indicate that the primary target of amyloid-beta (Abeta) peptide is the cellular membrane of neurons. In the context of these observations we investigated, using X-ray diffraction techniques, whether Abeta-metal complexes were able to affect lipid bilayers as a model of cell membranes. The binding of Al to Abeta gave particular conformational properties to the peptide that led to a marked alteration of the lipid bilayer representing phospholipids located in the outer monolayer of cell membranes. This effect was peculiar, since in our experimental conditions Abeta alone did not affect the lipid architecture, whereas the Al salt did, but only at concentrations several orders of magnitude higher than those of the Abeta-Al complex. In accordance with the effects observed with lipid bilayers, studies with human neuroblastoma cells demonstrated an impairment of cell functioning only in the presence of Abeta-Al complex. Our findings imply that Al, compared to the other Abeta-metal complexes tested, could have a specifically relevant effect in enhancing Abeta toxicity.

Shifting balance from neurodegeneration to regeneration of the brain: a novel therapeutic approach to Alzheimer's disease and related neurodegenerative conditions.

Neurodegeneration is one of the biggest public health problems in modern society. Age-associated neurodegeneration, which is accelerated several-fold in Alzheimers disease (AD) alone, is not only an enormous social and economic burden to the affected individuals and their families, but is also a great scientific challenge. Currently 25–35 million people worldwide suffer from AD, the single largest cause of dementia in middle- to old-aged individuals. These numbers are projected to triple by 2050 if no treatment to prevent or reverse AD is developed. The two histopathological hallmarks of AD and adults with Down syndrome, who develop AD histopathology in the fourth decade of their lives, are the intraneuronal neurofibrillary tangles and the extracellular Aβ plaques. The tangles in AD as well as in related neurodegenerative diseases called tauopathies are made up of microtubule associated protein tau modified by its abnormal hyperphosphorylation (Grundke-Iqbal et al., 1986a, b). The major components of plaques are 39–43 amino acid fragments, mostly Aβ1–40 and Aβ1–42 of β-amyloid precursor protein (Glenner and Wong, 1984). Ever since the discoveries of the composition of Aβ plaques and neurofibrillary tangles and implication of those lesions in neurodegeneration, most of the research in the AD field has been focused on inhibition of neurodegeneration by prevention or clearance of these lesions. However, to date, these efforts have not resulted in development of any disease-modifying drug (Iqbal and Grundke-Iqbal, 2010). Loss of neuronal plasticity and unsuccessful neurogenesis Both in AD and in its animal models the loss of neuronal plasticity is known to precede any overt formation of Aβ plaques and hyperphosphorylated (p) tau neurofibrillary tangles (Figure 1). Furthermore, the AD brain, in which the hippocampus is the most affected area of the brain and an area of the human brain where neurogenesis is known to occur throughout life, responds to neurodegeneration by stimulating neurogenesis (Figure 2). However, because of the lack of a proper neurotrophic microenvironment of the hippocampus this effort of the AD brain to replace lost neurons with new is unsuccessful (Li et al., 2008). Thus, one potential rational therapeutic approach to AD and other neurodegenerative conditions is to provide a neurotrophic environment in the brain that can materialize into successful neurogenesis and rescue neuronal plasticity deficit. Figure 1 Major features of Alzheimers disease pathology. Figure 2 Unsuccessful neurogenesis in Alzheimers disease. Pharmacologic rescue of neurogenesis and neuronal plasticity deficits and cognitive impairment Taking advantage of the findings that the hippocampal level of the mitogenic factor fibroblast growth factor-2 (FGF-2) is elevated in AD and the ciliary neurotrophic factor (CNTF) can counteract this effect (Chen et al., 2007), we developed a CNTF peptidergic compound, P6 (Chohan et al., 2011). Employing P6 and its more druggable form, P021, a tetrapeptide (DGGL) to which adamantylated glycine was added at the C-terminus, we studied dentate gyrus neurogenesis, neuronal plasticity and cognitive performance in aged Fisher rats and in a transgenic mouse model of both familial AD and familial frontotemporal dementia, the 3xTg-AD mouse model. The aged rat study was reported by Bolognin et al. (2014) and the 3xTg-AD studies by Blanchard et al. (2010) and Kazim et al. (2014). The Fisher rats are known to become cognitively impaired in old age. We administered P021 by gavage, 500 nmol (289.15 μg)/kg body weight daily for 3 months to 19–21 month old female Fisher rats. Another group of age-matched female rats was treated identically but with vehicle (saline) only and used as a vehicle-treated control. A group of 2–3 month old female rats treated with vehicle only served as young age controls. During the last week (12th week) of treatment all three groups of animals were tested for spatial reference memory by Morris Water Maze Task, following which the animals were sacrificed and their brains used for immunohistochemical and biochemical studies. We found that the aged rats were impaired in spatial memory as compared with young adult animals and P021 treatment could rescue this impairment. Immunohistochemical and biochemical analyses showed (i) a marked deficit in dentate gyrus neurogenesis in the aged rats which was partly rescued in P021-treated animals; (ii) P021 treatment increased both mRNA and protein expressions of BDNF in the hippocampi of aged rats; (iii) the aged rats displayed a decrease in phosphorylation of CREB which was rescued by P021 treatment; and (iv) P021 treatment increased the levels of synaptic markers, synaptophysin, synapsin-1 and glutamate receptors 2–3, and dendritic marker MAP2 in the aged rats. Magnetic resonance spectroscopy of the animals before perfusion showed an increase in the production of myoinositol which was rescued in the P021-treated animals. Thus, these studies collectively suggest that pharmacologic enhancement of neurogenesis and neuronal plasticity with a neurotrophic compound is a potential therapeutic approach to cognitive aging and AD. Beneficial effect of pharmacologic neuroregeneration on AD pathology in a transgenic mouse model In order to determine whether enhancement of regeneration of the brain can be potentially therapeutic both prior to and during the occurrence of AD histopathology, we employed the transgenic 3xTg-AD mouse model. This mouse model expresses human APPSWE and Presenilin-1 M146V knock-in AD and tau P301L frontotemporal dementia mutations, thus a model of both familial AD and familial frontotemporal dementia. These animals show cognitive impairment as early as at ~3 months of age, plaques at about 9–10 months of age and neurofibrillary tangles at around 12 months (Oddo et al., 2003). In one study (Blanchard et al., 2010), we administered intraperitoneally P6, 50 nmoles/mouse/day, for six weeks to 6–7 month old female 3xTg-AD mice and genetic background-matched wild-type mice. Mice treated identically to the above except with vehicle (saline) only provided additional controls. In a test for short term memory, the object recognition task, 3xTg-AD mice displayed impairment to discriminate between a familiar and a new object, accounting for short term memory deficit. Treatment with P6 reversed this impairment. Similarly in a spatial reference memory task employing water maze, 3xTg-AD mice presented delayed performance for learning compared to WT controls, but treatment with P6 reversed this impairment. Treatment with P6 did not induce any side effects as determined by body weight, anxiety, locomotive activity and coordination, and neophobia. The 3xTg-AD mice revealed a marked deficit in the dentate gyrus neurogenesis which was rescued by P6 treatment. The P6 treatment also rescued deficits in markers of neuronal plasticity, i.e., MAP2 and synaptophysin in 3xTg-AD mice. In a subsequent study (Kazim et al., 2014. Neurobiol. Dis. In press.) we investigated the effect of 6–12 months treatment with P021 on 9–10-month-old female 3xTg-AD mice. In this study we administered P021, 60 nmol/g diet up to 12 months. Control animals received the same diet but lacking P021. As in the above study with P6, the study with P021 also showed rescue of dentate gyrus neurogenesis and neuronal plasticity deficits and of cognitive impairment in 3xTg-AD mice. Moreover, the P021 treatment markedly reduced tau pathology and attenuated the generation but not the clearance of Aβ in 3xTg-AD mice. Similar to the above described studies, we have also reported rescue of cognitive impairment and neuroplasticity and neurogenesis deficits in an experimental rat model of sporadic AD (Bolognin et al., 2012) and in a trisomic Ts65Dn mouse model of Down syndrome (Blanchard et al., 2011). Collectively all these studies have established the proof of principle that shifting balance from neurodegeneration to regeneration of the brain with neurotrophic compounds can rescue Alzheimer type cognitive impairment and several features of the underlying pathology (Figure 3). Figure 3 Two major therapeutic approaches to Alzheimers disease and related conditions.

Animal Models of the Sporadic Form of Alzheimer's Disease: Focus on the Disease and Not Just the Lesions

Alzheimers disease is multifactorial and involves several different mechanisms. The sporadic form of the disease accounts for over 99% of the cases. As of yet, there is no practical and widely available animal model of the sporadic form of the disease. In the Alzheimers disease brain, the lysosomal enzyme asparaginyl endopeptidase is activated and translocated from the neuronal lysosomes to the cytoplasm, probably due to brain acidosis caused by ischemic changes associated with age-associated microinfarcts. The activated asparaginyl endopeptidase cleaves inhibitor-2 of protein phosphatase-2A, I2(PP2A), into I(2NTF) and I(2CTF) which translocate to the neuronal cytoplasm and inhibit the protein phosphatase activity and consequently the abnormal hyperphosphorylation of tau. Employing adeno-associated virus serotype 1 (AAV1) vector containing I(2NTF-CTF) and transduction of the brains of newborn rat pups with this virus, an animal model has been generated. The AAV1-I(2NTF-CTF) rats show neurodegeneration and cognitive impairment at 4 months and abnormal hyperphosphorylation and aggregation of tau and intraneuronal accumulation of amyloid-β at 13 months. The AAV1-I(2NTF-CTF) rats not only offer a disease-relevant model of the sporadic form of Alzheimers disease but also represent a practical and widely available animal model. This short perspective on the need to focus on and develop the disease-relevant models of the sporadic form of Alzheimers disease very much reflects the thinking of Inge Grundke-Iqbal who passed away on September 22, 2012 and to whom this article is dedicated.

Elevated Tau Level in Aged Rat Cerebrospinal Fluid Reduced by Treatment with a Neurotrophic Compound.

Alzheimers disease (AD) is the single major cause of dementia in middle- to old-age individuals, and, as of yet, no disease-modifying therapeutic drug is available for its treatment. A major obstacle in the successful development of disease-modifying therapeutic drugs has been the lack of suitable animal models of the sporadic form of AD as well as a biomarker that can be used both for therapeutic preclinical studies and for human clinical trials. Previously we showed neurogenesis and neuronal plasticity deficits and cognitive impairment and their rescue with a neurotrophic peptidergic compound, DGGLAG named P021, in aged Fisher rats. Here we show that P021 is blood-brain-barrier-permeable, and chronic oral treatment with this compound can reduce the brain level of total tau in the aged rats. Furthermore, cerebrospinal fluid (CSF) levels of both tau and Aβ/AβPP are elevated in the aged animals, and chronic treatment with P021 can reduce tau but not Aβ/AβPP to that of the levels found in young adult rats. Importantly, P021 does not induce any detectable immune reaction in rats. Collectively, these studies show the therapeutic potential of P021 as a disease-modifying compound and the suitability of the aged Fisher rats as a model of cerebral aging in which the therapeutic efficacy of a tau-reducing compound can be monitored in the CSF.

In Vivo Phenotyping Of Parkinson-Specific Stem Cells Reveals Increased a-Synuclein Levels But No Spreading

Parkinson′s disease is a progressive age-associated neurological disorder. One of the major neuropathological hallmarks of Parkinson’s disease is the appearance of protein aggregates, mainly consisting of the protein alpha-Synuclein. These aggregates have been described both in genetic as well as idiopathic forms of the disease. Currently, Parkinson’s disease patient-specific induced pluripotent stem cells (iPSCs) are mainly used for in vitro disease modeling or for experimental cell replacement approaches. Here, we demonstrate that these cells can be used for in vivo disease modeling. We show that Parkinson’s disease patient-specific, iPSC-derived neurons carrying the LRRK2-G2019S mutation show an upregulation of alpha-Synuclein after transplantation in the mouse brain. However, further investigations indicate that the increased human alpha-Synuclein levels fail to induce spreading or aggregation in the mouse brain. We therefore conclude that grafting of these cells into the mouse brain is suitable for cell autonomous in vivo disease modeling but has strong limitations beyond that. Furthermore, our results support the hypothesis that there might be a species barrier between human to mouse concerning alpha-Synuclein spreading.

A novel approach to derive human midbrain-specific organoids from neuroepithelial stem cells

Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Furthermore, animal models of human neurodegenerative diseases have failed dramatically, and the success rate of clinical trials based on these models has been disappointing. Here, we describe a novel and robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model to study Parkinson’s disease. Midbrain organoids are characterized in detail for neuronal, astroglial and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.