Persistent microglial activation and synaptic loss with behavioral abnormalities in mouse offspring exposed to CASPR2-antibodies in utero.

Gestational transfer of maternal antibodies against fetal neuronal proteins may be relevant to some neurodevelopmental disorders, but until recently there were no proteins identified. We recently reported a fivefold increase in CASPR2-antibodies in mid-gestation sera from mothers of children with intellectual and motor disabilities. Here, we exposed mice in utero to purified IgG from patients with CASPR2-antibodies (CASPR2-IgGs) or from healthy controls (HC-IgGs). CASPR2-IgG but not HC-IgG bound to fetal brain parenchyma, from which CASPR2-antibodies could be eluted. CASPR2-IgG exposed neonates achieved milestones similarly to HC-IgG exposed controls but, when adult, the CASPR2-IgG exposed progeny showed marked social interaction deficits, abnormally located glutamatergic neurons in layers V-VI of the somatosensory cortex, a 16% increase in activated microglia, and a 15-52% decrease in glutamatergic synapses in layers of the prefrontal and somatosensory cortices. Thus, in utero exposure to CASPR2-antibodies led to permanent behavioral, cellular, and synaptic abnormalities. These findings support a pathogenic role for maternal antibodies in human neurodevelopmental conditions, and CASPR2 as a potential target.

Further characterisation of the LPS model of Parkinson's disease: a comparison of intra-nigral and intra-striatal lipopolysaccharide administration on motor function, microgliosis and nigrostriatal neurodegeneration in the rat.

Chronic neuroinflammation has been established as one of the many processes involved in the pathogenesis of Parkinson's disease (PD). Because of this, researchers have attempted to replicate this pathogenic feature in animal models using the potent inflammagen, lipopolysaccharide (LPS), in order to gain better understanding of immune-mediated events in PD. However, although the effect of intra-cerebral LPS on neuroinflammation and neurodegeneration has been relatively well characterised, its impact on motor function has been less well studied. Therefore, the aim of this study was to further characterise the neuropathological and behavioural impact of intra-nigral and intra-striatal administration of LPS. To do, LPS (10 µg) or vehicle (sterile saline) were stereotaxically injected into the adult rat substantia nigra or striatum on one side only. The effect of LPS administration on lateralised motor function was assessed using the Corridor, Stepping and Whisker tests for two weeks post-injection, after which, amphetamine-induced rotational asymmetry was completed. Post-mortem, the impact of LPS on nigrostriatal degeneration and microgliosis was assessed using quantitative tyrosine hydroxylase and OX-42 immunohistochemistry respectively. We found that intra-nigral administration of LPS led to localised microgliosis in the substantia nigra and this was accompanied by nigrostriatal neurodegeneration and stable spontaneous motor deficits. In contrast, intra-striatal administration of LPS led to localised microgliosis in the striatum but this did not lead to any nigrostriatal neurodegeneration and only induced transient motor dysfunction. In conclusion, this study reveals the impact of intra-cerebral LPS administration on PD-related neuropathology and motor function, and it indicates that the intra-nigral model may be a highly relevant model as it is associated with stable motor decline underpinned by nigral microgliosis and nigrostriatal neurodegeneration.

Neuronal antibody prevalence in children with seizures under 3 years: A prospective national cohort.

OBJECTIVE: To report the prevalence of anti-neuronal antibodies in a prospective whole-nation cohort of children presenting with seizures before their third birthday. METHODS: This was a prospective population-based national cohort study involving all children presenting with new-onset epilepsy or complex febrile seizures before their third birthday over a 3-year period. Patients with previously identified structural, metabolic, or infectious cause for seizures were excluded. Serum samples were obtained at first presentation and tested for 7 neuronal antibodies using live cell-based assays. Clinical data were collected with structured proformas at recruitment and 24 months after presentation. In addition, patients with seizures and clinically suspected autoimmune encephalitis were independently identified by a review of the case records of all children <3 years of age in Scotland who had undergone EEG. RESULTS: Two hundred ninety-eight patients were identified and recruited and underwent autoantibody testing. Antibody positivity was identified in 18 of 298 (6.0%). The antibodies identified were GABA receptor B (n = 8, 2.7%), contactin-associated protein 2 (n = 4, 1.3%), glycine receptor (n = 3, 1.0%), leucine-rich glioma inactivated 1 (n = 2, 0.7%), NMDA receptor (n = 1, 0.3%), and GABA receptor A (n = 1, 0.3%). None of these patients had a clinical picture of autoimmune encephalitis. Seizure classification and clinical phenotype did not correlate with antibody positivity. CONCLUSIONS: Autoimmune encephalitis is very rare in early childhood. However serum neuronal antibodies are identified in 6.4% of children presenting with seizures at <3 years of age. Antibody testing should not be a routine clinical test in early childhood-onset epilepsy because, in the absence of other features of autoimmune encephalitis, antibody positivity is of doubtful clinical significance. Antibody testing should be reserved for patients with additional features of encephalitis.

Further validation of the corridor task for assessing deficit and recovery in the hemi-Parkinsonian rat: restoration of bilateral food retrieval by dopamine receptor agonism.

The corridor test is a newly developed test of sensorimotor integration that depends on a rat's ability to retrieve food from either side of its body. Rats with unilateral dopamine-depleting lesions neglect food on the contralateral side of their bodies, and selectively retrieve from the ipsilateral side. In the present study, the time-course for development of this deficit after injection of 6-hydroxydopamine into the striatum is determined using the corridor test. The ability of the dopamine receptor agonist, apomorphine, to reverse this impairment is also assessed. Lesioned rats developed an impairment in contralateral retrieval that was evident within a day (and stable for up to 2 weeks) after lesion surgery. Systemic injection of apomorphine significantly ameliorated this deficit, and restored the rats' ability to collect food from both sides of their bodies. This study confirms that the corridor test is highly sensitive to dysfunction of the nigrostriatal dopamine system, and suggests that it might be a useful tool for screening pharmacological approaches to the treatment of Parkinson's disease.

Fibrin As a Scaffold for Delivery of GDNF Overexpressing Stem Cells to the Adult Rat Brain.

Treatment of neurodegenerative disease is entering a new era where direct intracerebral delivery of therapeutic factors aims to restore normality to dysfunctional circuits. Cell-based therapeutic approaches, where virally manipulated mesenchymal stem cells (MSCs) overexpressing glial cell line derived neurotrophic factor (GDNF) are utilized as vehicles to deliver neurotrophic support to the Parkinsonian brain, have shown promising preclinical results at preserving dopaminergic neuron integrity. However, poor cell survival following transplantation will hinder clinical progression. One approach to improve MSCs survival following transplantation is to couple the cell engraftment procedure with a scaffold thereby providing a physical substrate upon which to eventually complex pro-survival factors. Evaluation of commercially available, clinically accepted materials with an established safety profile will expedite clinical translation. Therefore, this study sought to determine if a clinically used fibrin scaffold can be utilized as an adjunct to intracerebral cell transplantation without evoking an adverse host or stem cell response. Sixteen male Sprague-Dawley rats received bilateral intrastriatal transplants of 30 000 GDNF-transduced MSCs delivered in either control transplantation medium or a fibrin scaffold. Rats were sacrificed 1, 4, 7, and 14 days post-transplantation. Brains were analyzed to determine in situ polymerization and biodegradability of the fibrin scaffold, GDNF release from transplanted GDNF-MSCs, survival of the GDNF-MSC graft and the host's immune response to the transplant. This study found that fibrin scaffold was adaptable to intracerebral delivery with successful polymerization of the fibrin scaffold in situ. Inclusion of the fibrin scaffold was not detrimental to cell survival nor did it impede neurotrophin release from entrapped cells. Importantly, the inclusion of the fibrin scaffold was associated with a reduced host astroglial and microglial response compared to cells alone indicative of a favorable biocompatibility profile. Overall, fibrin represents an adaptable scaffold for inclusion in a minimally invasive cell-based therapeutic approach for neurodegenerative diseases.

Heat shock protein 70 reduces α-synuclein-induced predegenerative neuronal dystrophy in the α-synuclein viral gene transfer rat model of Parkinson's disease.

AIMS: It has become increasingly evident that the nigrostriatal degeneration associated with Parkinson's disease initiates at the level of the axonal terminals in the putamen, and this nigrostriatal terminal dystrophy is either caused or exacerbated by the presence of α-synuclein immunopositive neuronal inclusions. Therefore, strategies aimed at reducing α-synuclein-induced early neuronal dystrophy may slow or halt the progression to overt nigrostriatal neurodegeneration. Thus, this study sought to determine if adeno-associated virus (AAV) mediated overexpression of two molecular chaperone heat shock proteins, namely Hsp27 or Hsp70, in the AAV-α-synuclein viral gene transfer rat model of Parkinson's disease could prevent α-synuclein-induced early neuronal pathology. METHODS: Male Sprague-Dawley rats were intranigrally coinjected with pathogenic (AAV-α-synuclein) and putative therapeutic (AAV-Hsp27 or AAV-Hsp70) viral vectors and were sacrificed 18 weeks postviral injection. RESULTS: Intranigral injection of AAV-α-synuclein resulted in significant α-synuclein accumulation in the substantia nigra and striatal terminals which led to significant dystrophy of nigrostriatal dopaminergic neurons without overt nigrostriatal neurodegeneration. Coinjection of AAV-Hsp70, but not AAV-Hsp27, significantly reduced AAV-α-synuclein-induced neuronal dystrophy. CONCLUSIONS: These data confirm that overexpression of Hsp70 holds significant potential as a disease-modulating therapeutic approach for Parkinson's disease, with protective effects against early-onset α-synuclein-induced pathology demonstrated in the AAV-α-synuclein model.

The reduction in immunogenicity of neurotrophin overexpressing stem cells after intra-striatal transplantation by encapsulation in an in situ gelling collagen hydrogel.

Delivery of neurotrophic factors to the brain via genetically modified bone marrow-derived mesenchymal stem cells (MSCs) offers a promising neuroprotective strategy for neurodegenerative diseases. However, MSCs delivered to the CNS typically show poor survival post-transplantation, which is accompanied by microglial activation and astrocyte recruitment at the graft site. Recent studies have shown the potential of biomaterials to provide a supportive matrix for transplanted cells which may assist in the grafting process. In this study, an in situ gelling type I collagen hydrogel was evaluated as an intracerebral transplantation matrix for delivery of glial cell line-derived neurotrophic factor (GDNF)-overexpressing MSCs to the rat brain (GDNF-MSCs). In vitro analyses demonstrated that this collagen hydrogel did not affect the viability of the GDNF-MSCs nor did it prevent GDNF secretion into the surrounding medium. In vivo analyses also confirmed that the collagen hydrogel did not negatively impact on the survival of the cells and permitted GDNF secretion into the striatal parenchyma. Importantly, this study also revealed that transplanting GDNF-MSCs in a collagen hydrogel significantly diminished the host brain's response to the cells by reducing the recruitment of both microglia and astrocytes at the site of delivery. In conclusion, this hydrogel, which is composed of the natural extracellular matrix, collagen, was shown to be a well-tolerated cell delivery platform technology which could be functionalised to further aid cell support and graft integration.

The neurotoxicity of gene vectors and its amelioration by packaging with collagen hollow spheres.

Over the last twenty years there have been several reports on the use of nonviral vectors to facilitate gene transfer in the mammalian brain. Whilst a large emphasis has been placed on vector transfection efficiency, the study of the adverse effects upon the brain, caused by the vectors themselves, remains completely overshadowed. To this end, a study was undertaken to study the tissue response to three commercially available transfection agents in the brain of adult Sprague Dawley rats. The response to these transfection agents was compared to adeno-associated viral vector (AAV), PBS and naked DNA. Furthermore, the use of a collagen hollow sphere (CHS) sustained delivery system was analysed for its ability to reduce striatal toxicity of the most predominantly studied polymer vector, polyethyleneimine (PEI). The size of the gross tissue loss at the injection site was analysed after immunohistochemical staining and was used as an indication of acute toxicity. Polymeric vectors showed similar levels of acute brain toxicity as seen with AAV, and CHS were able to significantly reduce the toxicity of the PEI vector. In addition; the host response to the vectors was measured in terms of reactive astrocytes and microglial cell recruitment. To understand whether this gross tissue loss was caused by the direct toxicity of the vectors themselves an in vitro study on primary astrocytes was conducted. All vectors reduced the viability of the cells which is brought about by direct necrosis and apoptosis. The CHS delivery system reduced cell necrosis in the early stages of post administration. In conclusion, whilst polymeric gene vectors cause acute necrosis, administration in the brain causes adverse effects no worse than that of an AAV vector. Furthermore, packaging the PEI vector with CHS reduces surface charge and direct toxicity without elevating the host response.

Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells.

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.

Potential of rat bone marrow-derived mesenchymal stem cells as vehicles for delivery of neurotrophins to the Parkinsonian rat brain.

Issues related to the intra-cerebral delivery of glial cell line-derived neurotrophic factor (GDNF) have hampered its progression as a neuroprotective therapy for Parkinson's disease. Ex vivo gene therapy, where cells are virally transduced in vitro to produce a specific protein, may circumvent some of the problems associated with direct delivery of this neurotrophin to the brain. In this regard, bone marrow-derived mesenchymal stem cells (MSCs) offer an ideal cell source for ex vivo gene therapy because they are easily isolated from autologous sources, they are amenable to viral transduction and expansion in vitro, and they are hypoimmunogenic and non-tumourigenic in the brain. Thus the aim of this study was to determine the neurotrophic capacity of GDNF-transduced MSCs in a rat model of Parkinson's disease. Rats received intrastriatal transplants of GDNF-transduced MSCs 4days prior to induction of an intrastriatal 6-hydroxydopamine lesion. Quantitative tyrosine hydroxylase immunohistochemical staining revealed that GDNF-transduced MSCs were capable of inducing a pronounced local trophic effect in the denervated striatum which was evident by sprouting from the remaining dopaminergic terminals towards the neurotrophic milieu created by the transplanted cells. This strengthens the candidacy of MSCs as vehicles to deliver neurotrophins to the Parkinsonian brain.

Survival and immunogenicity of mesenchymal stem cells from the green fluorescent protein transgenic rat in the adult rat brain.

BACKGROUND: A major technical limitation in preclinical cell replacement research is the ability to discriminate between donor and host tissue after transplantation. This problem has been lessened by the availability of transgenic animals that express "reporter" genes, such as green fluorescent protein (GFP). OBJECTIVE: We determined the usefulness of one such transgenic reporter rat to assess the survival of bone marrow-derived rat mesenchymal stem cells (MSCs) following direct transplantation into the intact adult brain. We also sought to determine if the expression of GFP in the brain affected the survival of the MSCs or the host's neuroimmune response to the cells. METHODS: Rats received intrastriatal injections of sterile transplantation medium, 100 000 normal MSCs, or 100 000 GFP-MSCs and were killed humanely 1, 4, 7, 28, and 42 days posttransplantation for astrocyte and microglial immunohistochemical staining. RESULTS: GFP-MSCs were evident at each examination, although their survival declined over time. Graft volume estimates comparing normal and GFP-MSCs revealed that GFP expression did not adversely affect the survival of the stem cells in the brain. Furthermore, immunostaining for astrocytes and microglia revealed that expression of the reporter protein did not affect the immunogenicity of the stem cells. CONCLUSIONS: These data indicate the usefulness of GFP for investigating the survival of MSCs following transplantation to the brain. However, the mechanisms responsible for the poor survival of the stem cells must be elucidated if these cells are to serve cell-based therapies for neurodegenerative disorders.