Bilateral Ischial Tuberosity Stress Fractures in an Adolescent Football Player: A Case Report.

ABSTRACT: Pelvic stress fractures are rare, making up an estimated 1% to 7% of all stress fractures with the primary locations being the pubic rami, pubic symphysis, and sacrum. Two cases of stress fractures of the ischium have been previously described in the literature, with both occurring in the ischial body. In this case, a 17-year-old high school American football player presented with nonspecific pelvic pain and bilateral point tenderness on deep palpation of the ischial tuberosities. Advanced imaging identified bilateral ischial tuberosity stress fractures. This report outlines the diagnosis and management of the first reported case of bilateral ischial tuberosity stress fractures. We report how ischial tuberosity stress fractures present clinically, potential management strategies, and highlight the use of computed tomography imaging for pelvic stress fractures. Knowledge of unusual stress fracture locations may improve early diagnosis, limit complications, reduce healthcare costs, and promote an accelerated recovery time.

Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols.

An anti-neuroinflammatory that targets dysregulated glia enhances the efficacy of CNS-directed gene therapy in murine infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative lysosomal storage disease (LSD) caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). Studies in Ppt1(-/-) mice demonstrate that glial activation is central to the pathogenesis of INCL. Astrocyte activation precedes neuronal loss, while cytokine upregulation associated with microglial reactivity occurs before and concurrent with neurodegeneration. Therefore, we hypothesized that cytokine cascades associated with neuroinflammation are important therapeutic targets for the treatment of INCL. MW01-2-151SRM (MW151) is a blood-brain barrier penetrant, small-molecule anti-neuroinflammatory that attenuates glial cytokine upregulation in models of neuroinflammation such as traumatic brain injury, Alzheimer's disease, and kainic acid toxicity. Thus, we used MW151, alone and in combination with CNS-directed, AAV-mediated gene therapy, as a possible treatment for INCL. MW151 alone decreased seizure susceptibility. When combined with AAV-mediated gene therapy, treated INCL mice had increased life spans, improved motor performance, and eradication of seizures. Combination-treated INCL mice also had decreased brain atrophy, astrocytosis, and microglial activation, as well as intermediary effects on cytokine upregulation. These data suggest that MW151 can attenuate seizure susceptibility but is most effective when used in conjunction with a therapy that targets the primary genetic defect.

Nonclinical evaluation of CNS-administered TPP1 enzyme replacement in canine CLN2 neuronal ceroid lipofuscinosis.

The CLN2 form of neuronal ceroid lipofuscinosis, a type of Batten disease, is a lysosomal storage disorder caused by a deficiency of the enzyme tripeptidyl peptidase-1 (TPP1). Patients exhibit progressive neurodegeneration and loss of motor, cognitive, and visual functions, leading to death by the early teenage years. TPP1-null Dachshunds recapitulate human CLN2 disease. To characterize the safety and pharmacology of recombinant human (rh) TPP1 administration to the cerebrospinal fluid (CSF) as a potential enzyme replacement therapy (ERT) for CLN2 disease, TPP1-null and wild-type (WT) Dachshunds were given repeated intracerebroventricular (ICV) infusions and the pharmacokinetic (PK) profile, central nervous system (CNS) distribution, and safety were evaluated. TPP1-null animals and WT controls received 4 or 16mg of rhTPP1 or artificial cerebrospinal fluid (aCSF) vehicle every other week. Elevated CSF TPP1 concentrations were observed for 2-3 days after the first ICV infusion and were approximately 1000-fold higher than plasma levels at the same time points. Anti-rhTPP1 antibodies were detected in CSF and plasma after repeat rhTPP1 administration, with titers generally higher in TPP1-null than in WT animals. Widespread brain distribution of rhTPP1 was observed after chronic administration. Expected histological changes were present due to the CNS delivery catheters and were similar in rhTPP1 and vehicle-treated animals, regardless of genotype. Neuropathological evaluation demonstrated the clearance of lysosomal storage, preservation of neuronal morphology, and reduction in brain inflammation with treatment. This study demonstrates the favorable safety and pharmacology profile of rhTPP1 ERT administered directly to the CNS and supports clinical evaluation in patients with CLN2 disease.

Appalachian Nursing Homes: A Descriptive Analysis of Quality, Staffing, and Cost.

OBJECTIVE: To examine if the quality of care in Appalachian nursing homes in metropolitan, micropolitan, and rural areas differs from those in non-Appalachian regions of the United States. DESIGN: Retrospective analysis of Medicare Cost Reports, combined with data from Nursing Home Compare, LTCFocus, and Medicare, Post-Acute Care, and Hospice Public Use Form. Nursing homes were classified using Rural-Urban Commuting Area Codes. SETTING AND PARTICIPANTS: Data from 14,040 nursing homes reporting on staffing, costs, and quality of care metrics from 2013 to 2019 were analyzed. METHODS: Descriptive analyses compared resident and facility characteristics, quality, staffing, and cost outcomes between non-Appalachian and Appalachian nursing homes in metropolitan, micropolitan, and rural areas. Regressions compared quality, staffing, and cost outcomes among (1) Appalachian metropolitan and non-Appalachian nursing homes, (2) Appalachian micropolitan and non-Appalachian nursing homes, and (3) Appalachian rural and non-Appalachian nursing homes. Outcomes included health deficiency index scores, Medicare spending per beneficiary, staffing hours per resident day (registered nurse, licensed practical nurse, certified nursing assistant per resident day), and 5 Minimum Data Set metrics for short-stay and long-stay residents. RESULTS: Appalachian nursing homes are more likely to be hospital-based, for-profit, multifacility chain affiliated, and have higher proportions of white and Medicaid residents. Regression analyses revealed that Appalachian metropolitan nursing homes have 3.3% fewer certified nursing assistant hours per resident day, a 16.5% higher health deficiency score index, and 4.2% higher Medicare spending per beneficiary compared with non-Appalachian homes. Appalachian micropolitan nursing homes showed 7.4% fewer registered nurse hours per resident day and 6.9% higher Medicare spending per beneficiary. Appalachian rural nursing homes had 16.7% more registered nurse hours per resident day, 22.7% lower health deficiency index scores, and 10.7% higher Medicare spending per beneficiary. Minimum Data Set measures varied, with Appalachian nursing homes performing better on some metrics and worse on others. CONCLUSIONS AND IMPLICATIONS: Appalachia lags behind in staffing and Medicare spending per beneficiary. These disparities should be considered by policymakers advocating for Appalachia's senior citizens.

Synergistic effects of central nervous system-directed gene therapy and bone marrow transplantation in the murine model of infantile neuronal ceroid lipofuscinosis.

OBJECTIVE: Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited childhood neurodegenerative disorder caused by the loss of palmitoyl protein thioesterase-1 (PPT1) activity. Affected children suffer from blindness, epilepsy, motor dysfunction, cognitive decline, and premature death. The Ppt1(-/-) mouse shares the histological and clinical features of INCL. Previous single-therapy approaches using small molecule drugs, gene therapy, or neuronal stem cells resulted in partial histological correction, with minimal improvements in motor function or lifespan. Here, we combined central nervous system (CNS)-directed adeno-associated virus (AAV)2/5-mediated gene therapy with bone marrow transplantation (BMT) in the INCL mouse. METHODS: At birth, Ppt1(-/-) and wild-type mice were given either intracranial injections of AAV2/5-PPT1 or bone marrow transplantation, separately as well as in combination. To assess function, we measured rotorod performance monthly as well as lifespan. At terminal time points, we evaluated the therapeutic effects on several INCL-specific parameters, such as cortical thickness, autofluorescent accumulation, and glial activation. Finally, we determined levels of PPT1 enzyme activity and bone marrow engraftment in treated mice. RESULTS: AAV2/5-mediated gene therapy alone resulted in significant histological correction, improved motor function, and increased lifespan. Interestingly, the addition of BMT further increased the lifespan of treated mice and led to dramatic, sustained improvements in motor function. These data are truly striking, given that BMT alone is ineffective, yet it synergizes with CNS-directed gene therapy to dramatically increase efficacy and lifespan. INTERPRETATION: AAV2/5-mediated gene therapy in combination with BMT provides an unprecedented increase in lifespan as well as dramatic improvement on functional and histological parameters.

Simultaneous Bilateral Quadriceps Tendon Rupture Secondary to Parathyroid Carcinoma: A Case Report.

Simultaneous bilateral quadriceps tendon ruptures are a rare occurrence commonly associated with a traumatic event or systemic disease. A 31-year-old man presented with simultaneous bilateral quadriceps tendon ruptures with associated hyperparathyroidism secondary to parathyroid carcinoma. The injury occurred after the patient attempted to lift a small wooden log from the ground. We discussed the multidisciplinary management of this patient resulting in bilateral quadriceps tendon repairs, tumor resection, and oncological and endocrinological restoration. Clinical follow-up is reported at 15 years after surgery. Parathyroid carcinoma is an extremely rare cancer and rarely the cause of hyperparathyroidism. The systemic effects of the tumor eventually lead to the rupturing of both quadriceps tendons. Orthopaedic physicians must remain vigilant in identifying the root cause of injuries that are atypical in nature.

Early glial activation, synaptic changes and axonal pathology in the thalamocortical system of Niemann-Pick type C1 mice.

Niemann-Pick disease type C (NPC) is an inherited lysosomal storage disease characterised by accumulation of cholesterol and glycosphingolipids. NPC patients suffer a progressive neurodegenerative phenotype presenting with motor dysfunction, mental retardation and cognitive decline. To examine the onset and progression of neuropathological insults in NPC we have systematically examined the CNS of a mouse model of NPC1 (Npc1(-/-) mice) at different stages of the disease course. This revealed a specific spatial and temporal pattern of neuropathology in Npc1(-/-) mice, highlighting that sensory thalamic pathways are particularly vulnerable to loss of NPC1 resulting in neurodegeneration in Npc1(-/-) mice. Examination of markers of astrocytosis and microglial activation revealed a particularly pronounced reactive gliosis in the thalamus early in the disease, which subsequently also occurred in interconnected cortical laminae at later ages. Our examination of the precise staging of events demonstrate that the relationship between glia and neurons varies between brain regions in Npc1(-/-) mice, suggesting that the cues causing glial reactivity may differ between brain regions. In addition, aggregations of pre-synaptic markers are apparent in white matter tracts and the thalamus and are likely to be formed within axonal spheroids. Our data provide a new perspective, revealing a number of events that occur prior to and alongside neuron loss and highlighting that these occur in a pathway dependent manner.

Intravenous high-dose enzyme replacement therapy with recombinant palmitoyl-protein thioesterase reduces visceral lysosomal storage and modestly prolongs survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis.

PPT1-related neuronal ceroid lipofuscinosis (NCL) is a lysosomal storage disorder caused by deficiency in a soluble lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1). Enzyme replacement therapy (ERT) has not been previously examined in a preclinical animal model. Homozygous PPT1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death by around 8 months. In the current study, PPT1 knockout mice were treated with purified recombinant PPT1 (0.3 mg, corresponding to 12 mg/kg or 180 U/kg for a 25 g mouse) administered intravenously weekly either 1) from birth; or 2) beginning at 8 weeks of age. The treatment was surprisingly well tolerated and neither anaphylaxis nor antibody formation was observed. In mice treated from birth, survival increased from 236 to 271 days (p<0.001) and the onset of motor deterioration was similarly delayed. In mice treated beginning at 8 weeks, no increases in survival or motor performance were seen. An improvement in neuropathology in the thalamus was seen at 3 months in mice treated from birth, and although this improvement persisted it was attenuated by 7 months. Outside the central nervous system, substantial clearance of autofluorescent storage material in many tissues was observed. Macrophages in spleen, liver and intestine were especially markedly improved, as were acinar cells of the pancreas and tubular cells of the kidney. These findings suggest that ERT may be an option for addressing visceral storage as part of a comprehensive approach to PPT1-related NCL, but more effective delivery methods to target the brain are needed.

Intravenous administration of AAV2/9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system.

Several diseases of the nervous system are characterized by neurodegeneration and death in childhood. Conventional medicine is ineffective, but fetal or neonatal gene therapy may provide an alternative route to treatment. We evaluated the ability of single-stranded and self-complementary adeno-associated virus pseudotype 2/9 (AAV2/9) to transduce the nervous system and target gene expression to specific neural cell types following intravenous injection into fetal and neonatal mice, using control uninjected age-matched mice. Fetal and neonatal administration produced global delivery to the central (brain, spinal cord, and all layers of the retina) and peripheral (myenteric plexus and innervating nerves) nervous system but with different expression profiles within the brain; fetal and neonatal administration resulted in expression in neurons and protoplasmic astrocytes, respectively. Neither single-stranded nor self-complementary AAV2/9 triggered a microglia-mediated immune response following either administration. In summary, intravenous AAV2/9 targets gene expression to specific neural cell types dependent on developmental stage. This represents a powerful tool for studying nervous system development and disease. Furthermore, it may provide a therapeutic strategy for treatment of early lethal genetic diseases, such as Gaucher disease, and for disabling neuropathies, such as preterm brain injury.

Combination small molecule PPT1 mimetic and CNS-directed gene therapy as a treatment for infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a profoundly neurodegenerative disease of children caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). There is currently no effective therapy for this invariably fatal disease. To date, preclinical experiments using single treatments have resulted in incremental clinical improvements. Therefore, we determined the efficacy of CNS-directed AAV2/5-mediated gene therapy alone and in combination with the systemic delivery of the lysosomotropic PPT1 mimetic phosphocysteamine. Since CNS-directed gene therapy provides relatively high levels of PPT1 activity to specific regions of the brain, we hypothesized that phosphocysteamine would complement that activity in regions expressing subtherapeutic levels of the enzyme. Results indicate that CNS-directed gene therapy alone provided the greatest improvements in biochemical and histological measures as well as motor function and life span. Phosphocysteamine alone resulted in only minor improvements in motor function and no increase in lifespan. Interestingly, phosphocysteamine did not increase the biochemical and histological response when combined with AAV2/5-mediated gene therapy, but it did result in an additional improvement in motor function. These data suggest that a CNS-directed gene therapy approach provides significant clinical benefit, and the addition of the small molecule PPT1 mimetic can further increase that response.

DHHC5 interacts with PDZ domain 3 of post-synaptic density-95 (PSD-95) protein and plays a role in learning and memory.

A family of integral membrane proteins containing a signature DHHC motif has been shown to display protein S-acyltransferase activity, modifying cysteine residues in proteins with fatty acids. The physiological roles of these proteins have largely been unexplored. Here we report that mice homozygous for a hypomorphic allele of a previously uncharacterized member, DHHC5, are born at half the expected rate, and survivors show a marked deficit in contextual fear conditioning, an indicator of defective hippocampal-dependent learning. DHHC5 is highly enriched in a post-synaptic density preparation and co-immunoprecipitates with post-synaptic density protein-95 (PSD-95), an interaction that is mediated through binding of the carboxyl terminus of DHHC5 and the PDZ3 domain of PSD-95. Immunohistochemistry demonstrated that DHHC5 is expressed in the CA3 and dentate gyrus in the hippocampus. These findings point to a previously unsuspected role for DHHC5 in post-synaptic function affecting learning and memory.

Molecular correlates of axonal and synaptic pathology in mouse models of Batten disease.

Neuronal ceroid lipofuscinoses (NCLs; Batten disease) are collectively the most frequent autosomal-recessive neurodegenerative disease of childhood, but the underlying cellular and molecular mechanisms remain unclear. Several lines of evidence have highlighted the important role that non-somatic compartments of neurons (axons and synapses) play in the instigation and progression of NCL pathogenesis. Here, we report a progressive breakdown of axons and synapses in the brains of two different mouse models of NCL: Ppt1(-/-) model of infantile NCL and Cln6(nclf) model of variant late-infantile NCL. Synaptic pathology was evident in the thalamus and cortex of these mice, but occurred much earlier within the thalamus. Quantitative comparisons of expression levels for a subset of proteins previously implicated in regulation of axonal and synaptic vulnerability revealed changes in proteins involved with synaptic function/stability and cell-cycle regulation in both strains of NCL mice. Protein expression changes were present at pre/early-symptomatic stages, occurring in advance of morphologically detectable synaptic or axonal pathology and again displayed regional selectivity, occurring first within the thalamus and only later in the cortex. Although significant differences in individual protein expression profiles existed between the two NCL models studied, 2 of the 15 proteins examined (VDAC1 and Pttg1) displayed robust and significant changes at pre/early-symptomatic time-points in both models. Our study demonstrates that synapses and axons are important early pathological targets in the NCLs and has identified two proteins, VDAC1 and Pttg1, with the potential for use as in vivo biomarkers of pre/early-symptomatic axonal and synaptic vulnerability in the NCLs.

Current therapies for the soluble lysosomal forms of neuronal ceroid lipofuscinosis.

The NCLs (neuronal ceroid lipofuscinoses) are the most common inherited paediatric neurodegenerative disorder. Although genetically distinct, NCLs can be broadly divided into two categories: one in which the mutation results in a defect in a transmembrane protein, and the other where the defect lies in a soluble lysosomal enzyme. A number of therapeutic approaches are applicable to the soluble lysosomal forms of NCL based on the phenomenon of cross-correction, whereby the ubiquitously expressed mannose 6-phosphate/IGF (insulin-like growth factor) II receptor provides an avenue for endocytosis, trafficking and lysosomal processing of extracellularly delivered enzyme. The present review discusses therapeutic utilization of cross-correction by enzyme-replacement therapy, gene therapy and stem cell therapy for the NCLs, along with an overview of the recent progress in translating these treatments into the clinic.

In utero gene transfer to the mouse nervous system.

The cellular and molecular environment present in the fetus and early newborn provides an excellent opportunity for effective gene transfer. Innate and pre-existing anti-vector immunity may be attenuated or absent and the adaptive immune system predisposed to tolerance towards xenoproteins. Stem cell and progenitor cell populations are abundant, active and accessible. In addition, for treatment of early lethal genetic diseases of the nervous system, the overarching advantage may be that early gene supplementation prevents the onset of irreversible pathological changes. Gene transfer to the fetal mouse nervous system was achieved, albeit inefficiently, as far back as the mid-1980s. Recently, improvements in vector design and production have culminated in near-complete correction of a mouse model of spinal muscular atrophy. In the present article, we review perinatal gene transfer from both a therapeutic and technological perspective.

Generation of light-producing somatic-transgenic mice using adeno-associated virus vectors.

We have previously designed a library of lentiviral vectors to generate somatic-transgenic rodents to monitor signalling pathways in diseased organs using whole-body bioluminescence imaging, in conscious, freely moving rodents. We have now expanded this technology to adeno-associated viral vectors. We first explored bio-distribution by assessing GFP expression after neonatal intravenous delivery of AAV8. We observed widespread gene expression in, central and peripheral nervous system, liver, kidney and skeletal muscle. Next, we selected a constitutive SFFV promoter and NFκB binding sequence for bioluminescence and biosensor evaluation. An intravenous injection of AAV8 containing firefly luciferase and eGFP under transcriptional control of either element resulted in strong and persistent widespread luciferase expression. A single dose of LPS-induced a 10-fold increase in luciferase expression in AAV8-NFκB mice and immunohistochemistry revealed GFP expression in cells of astrocytic and neuronal morphology. Importantly, whole-body bioluminescence persisted up to 240 days. We have validated a novel biosensor technology in an AAV system by using an NFκB response element and revealed its potential to monitor signalling pathway in a non-invasive manner in a model of LPS-induced inflammation. This technology complements existing germline-transgenic models and may be applicable to other rodent disease models.

Fetal gene therapy for neurodegenerative disease of infants.

For inherited genetic diseases, fetal gene therapy offers the potential of prophylaxis against early, irreversible and lethal pathological change. To explore this, we studied neuronopathic Gaucher disease (nGD), caused by mutations in GBA. In adult patients, the milder form presents with hepatomegaly, splenomegaly and occasional lung and bone disease; this is managed, symptomatically, by enzyme replacement therapy. The acute childhood lethal form of nGD is untreatable since enzyme cannot cross the blood-brain barrier. Patients with nGD exhibit signs consistent with hindbrain neurodegeneration, including neck hyperextension, strabismus and, often, fatal apnea1. We selected a mouse model of nGD carrying a loxP-flanked neomycin disruption of Gba plus Cre recombinase regulated by the keratinocyte-specific K14 promoter. Exclusive skin expression of Gba prevents fatal neonatal dehydration. Instead, mice develop fatal neurodegeneration within 15 days2. Using this model, fetal intracranial injection of adeno-associated virus (AAV) vector reconstituted neuronal glucocerebrosidase expression. Mice lived for up to at least 18 weeks, were fertile and fully mobile. Neurodegeneration was abolished and neuroinflammation ameliorated. Neonatal intervention also rescued mice but less effectively. As the next step to clinical translation, we also demonstrated the feasibility of ultrasound-guided global AAV gene transfer to fetal macaque brains.

Proteomic mapping of differentially vulnerable pre-synaptic populations identifies regulators of neuronal stability in vivo.

Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the Cln3 -/- mouse model of a juvenile form of NCL. We profiled and compared the molecular composition of anatomically-distinct, differentially-affected pre-synaptic populations from the Cln3 -/- mouse brain using proteomics followed by bioinformatic analyses. Identified protein candidates were then tested using a Drosophila CLN3 model to study their ability to modify the CLN3-neurodegenerative phenotype in vivo. We identified differential perturbations in a range of molecular cascades correlating with synaptic vulnerability, including valine catabolism and rho signalling pathways. Genetic and pharmacological targeting of key 'hub' proteins in such pathways was sufficient to modulate phenotypic presentation in a Drosophila CLN3 model. We propose that such a workflow provides a target rich method for the identification of novel disease regulators which could be applicable to the study of other conditions where appropriate models exist.

Glial cells are functionally impaired in juvenile neuronal ceroid lipofuscinosis and detrimental to neurons.

The neuronal ceroid lipofuscinoses (NCLs or Batten disease) are a group of inherited, fatal neurodegenerative disorders of childhood. In these disorders, glial (microglial and astrocyte) activation typically occurs early in disease progression and predicts where neuron loss subsequently occurs. We have found that in the most common juvenile form of NCL (CLN3 disease or JNCL) this glial response is less pronounced in both mouse models and human autopsy material, with the morphological transformation of both astrocytes and microglia severely attenuated or delayed. To investigate their properties, we isolated glia and neurons from Cln3-deficient mice and studied their basic biology in culture. Upon stimulation, both Cln3-deficient astrocytes and microglia also showed an attenuated ability to transform morphologically, and an altered protein secretion profile. These defects were more pronounced in astrocytes, including the reduced secretion of a range of neuroprotective factors, mitogens, chemokines and cytokines, in addition to impaired calcium signalling and glutamate clearance. Cln3-deficient neurons also displayed an abnormal organization of their neurites. Most importantly, using a co-culture system, Cln3-deficient astrocytes and microglia had a negative impact on the survival and morphology of both Cln3-deficient and wildtype neurons, but these effects were largely reversed by growing mutant neurons with healthy glia. These data provide evidence that CLN3 disease astrocytes are functionally compromised. Together with microglia, they may play an active role in neuron loss in this disorder and can be considered as potential targets for therapeutic interventions.

Neural stem cell grafts reduce the extent of neuronal damage in a mouse model of global ischaemia.

The therapeutic potential of neural stem cell transplantation has been well demonstrated in many models of focal brain damage. However, few studies have sought to determine whether neural stem cells are therapeutic in models of diffuse brain injury, such as observed in Alzheimer's disease and global ischaemia. The present study investigated the effects of transplanted MHP36 neural stem cells on the extent of ischaemic damage in a mouse model of global ischaemia and the effects of the immunosuppressive agent cyclosporin A (CsA). C57Bl/6J mice received an intrastriatal graft of MHP36 neural stem cells 3 days after selective neuronal damage had been induced by global ischaemia. The experimental group was subdivided into CsA or saline controls. We discovered that grafts of MHP36 neural stem cells were able to differentiate into neurons and reduce the extent of ischaemic neuronal damage. This reduction was particularly apparent at 4 week post-transplantation and is independent of CsA immunosuppression. MHP36 cells survived robustly in host ischaemic brain and migrated away from the injection tract towards the caudate nucleus and corpus callosum. Although MHP36 grafts were associated with an acute inflammatory response from reactive astrocytes and microglia at 1 week post-transplantation, this decreased markedly by 4 weeks post-transplantation even in the absence of CsA immunosuppression. This is the first study showing a therapeutic benefit of neural stem cells in a highly diffuse brain injury, further highlighting the possibilities of stem cell transplantation for all types of neurodegenerative disease.