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Abstract Wildfires in the southwestern United States are increasingly frequent and severe, but whether these trends exceed historical norms remains contested. Here we combine dendroecological records, satellite-derived burn severity, and field measured tree mortality to compare historical (1700-1880) and contemporary (1985-2020) fire regimes at tree-ring fire-scar sites in Arizona and New Mexico. We found that contemporary fire frequency, including recent, record fire years, is still

IntroductionClimate change is predicted to increase the frequency of extreme single-day fire spread events, with major ecological and social implications. In contrast with well-documented spatio-temporal patterns of wildfire ignitions and perimeters, daily progression remains poorly understood across continental spatial scales, particularly for extreme single-day events (“blow ups”). Here, we characterize daily wildfire spread across North America, including occurrence of extreme single-day events, duration and seasonality of fire and extremes, and ecoregional climatic niches of fire in terms of Actual Evapotranspiration (AET) and Climatic Water Deficit (CWD) annual climate normals.MethodsRemotely sensed daily progression of 9,636 wildfires ≥400 ha was used to characterize ecoregional patterns of fire growth, extreme single-day events, duration, and seasonality. To explore occurrence, extent, and impacts of single-day extremes among ecoregions, we considered complementary ecoregional and continental extreme thresholds (Ecoregional or Continental Mean Daily Area Burned + 2SD). Ecoregional spread rates were regressed against AET and CWD to explore climatic influence on spread.ResultsWe found three-fold differences in mean Daily Area Burned among 10 North American ecoregions, ranging from 260 ha day−1 in the Marine West Coast Forests to 751 ha day−1 in Mediterranean California. Ecoregional extreme thresholds ranged from 3,829 ha day−1 to 16,626 ha day−1, relative to a continental threshold of 7,173 ha day−1. The ~3% of events classified as extreme cumulatively account for 16–55% of total area burned among ecoregions. We observed four-fold differences in mean fire duration, ranging from 2.7 days in the Great Plains to 10.5 days in Northwestern Forested Mountains. Regions with shorter fire durations also had greater daily area burned, suggesting a paradigm of fast-growing short-duration fires in some regions and slow-growing long-duration fires elsewhere. CWD had a weak positive relationship with spread rate and extreme thresholds, and there was no pattern for AET.DiscussionRegions with shorter fire durations had greater daily area burned, suggesting a paradigm of fast-growing short-duration fires in some regions and slow-growing long-duration fires elsewhere. Although climatic conditions can set the stage for ignition and influence vegetation and fuels, finer-scale mechanisms likely drive variation in daily spread. Daily fire progression offers valuable insights into the regional and seasonal distributions of extreme single-day spread events, and how these events shape net fire effects.

Abstract The historical role of fire in sagebrush (Artemisia tridentata) landscapes remains poorly understood, yet is important to inform management and conservation of obligate species such as the threatened Gunnison Sage‐grouse (GUSG; Centrocercus minimus). We reconstructed fire histories from tree‐ring fire scars at sagebrush–forest ecotones (10 sites, 111 trees) to better understand the role of fire in sagebrush landscapes of the Upper Gunnison Basin (UGB), Colorado, and how fire may have changed following Euro‐American settlement. We assessed likely influences of historical fire by surveying plant composition and structure at 100 sagebrush sites with and without recent (2001–2020) fires. Tree‐ring fire scars revealed a history of repeated low‐severity fire at sagebrush–forest ecotones until 1892, followed by over a century without fire. Between 1684 and 1892, the mean fire interval (MFI) among sites averaged 49.6 years (ranging from 18.2 to 119 years). Fire over this period occurred synchronously at two or more sites on average every 23.6 years, potentially indicative of spread between sites. Most (70%) of the historical fires burned in the early growing season, consistent with times of strong wind. Recent burns exhibited reductions in sagebrush cover (5% vs. 25% in unburned sites) and concomitant increases in herbaceous cover (55% vs. 40%). These differences declined over time but persisted for at least two decades. Burned sites were dominated by native perennial grasses, forbs, and resprouting shrub species. Historically, such openings may have served as seasonal GUSG habitat. Our results indicate that parts of the UGB sagebrush landscapes were characterized historically by frequent fire and dynamic vegetation mosaics that included open, grassy patches. These findings support the use of prescribed fire to restore and maintain this ecological process and vegetation heterogeneity. However, the contemporary context for fire has changed and now includes substantially reduced, Endangered Species Act (ESA)‐listed GUSG populations, increased risk of non‐native plant invasion, and climate warming. These circumstances highlight new risks, information needs, and opportunities for key knowledge co‐production via management–research partnerships.

Abstract Fire regimes in North American forests are diverse and modern fire records are often too short to capture important patterns, trends, feedbacks, and drivers of variability. Tree‐ring fire scars provide valuable perspectives on fire regimes, including centuries‐long records of fire year, season, frequency, severity, and size. Here, we introduce the newly compiled North American tree‐ring fire‐scar network (NAFSN), which contains 2562 sites, >37,000 fire‐scarred trees, and covers large parts of North America. We investigate the NAFSN in terms of geography, sample depth, vegetation, topography, climate, and human land use. Fire scars are found in most ecoregions, from boreal forests in northern Alaska and Canada to subtropical forests in southern Florida and Mexico. The network includes 91 tree species, but is dominated by gymnosperms in the genus Pinus. Fire scars are found from sea level to >4000‐m elevation and across a range of topographic settings that vary by ecoregion. Multiple regions are densely sampled (e.g., >1000 fire‐scarred trees), enabling new spatial analyses such as reconstructions of area burned. To demonstrate the potential of the network, we compared the climate space of the NAFSN to those of modern fires and forests; the NAFSN spans a climate space largely representative of the forested areas in North America, with notable gaps in warmer tropical climates. Modern fires are burning in similar climate spaces as historical fires, but disproportionately in warmer regions compared to the historical record, possibly related to under‐sampling of warm subtropical forests or supporting observations of changing fire regimes. The historical influence of Indigenous and non‐Indigenous human land use on fire regimes varies in space and time. A 20th century fire deficit associated with human activities is evident in many regions, yet fire regimes characterized by frequent surface fires are still active in some areas (e.g., Mexico and the southeastern United States). These analyses provide a foundation and framework for future studies using the hundreds of thousands of annually‐ to sub‐annually‐resolved tree‐ring records of fire spanning centuries, which will further advance our understanding of the interactions among fire, climate, topography, vegetation, and humans across North America.

Abstract Altered fire regimes can drive major and enduring compositional shifts or losses of forest ecosystems. In western North America, ponderosa pine and dry mixed‐conifer forest types appear increasingly vulnerable to uncharacteristically extensive, high‐severity wildfire. However, unburned or only lightly impacted forest stands that persist within burn mosaics—termed fire refugia—may serve as tree seed sources and promote landscape recovery. We sampled tree regeneration along gradients of fire refugia proximity and density at 686 sites within the perimeters of 12 large wildfires that occurred between 2000 and 2005 in the interior western United States. We used generalized linear mixed‐effects models to elucidate statistical relationships between tree regeneration and refugia pattern, including a new metric that incorporates patch proximity and proportional abundance. These relationships were then used to develop a spatially explicit landscape simulation model. We found that regeneration by ponderosa pine and obligate‐seeding mixed‐conifer tree species assemblages was strongly and positively predicted by refugia proximity and density. Simulation models revealed that for any given proportion of the landscape occupied by refugia, small patches produced greater landscape recovery than large patches. These results highlight the disproportionate importance of small, isolated islands of surviving trees, which may not be detectable with coarse‐scale satellite imagery. Findings also illustrate the interplay between patch‐scale resistance and landscape‐scale resilience: Disturbance‐resistant settings (fire refugia) can entrain resilience (forest regeneration) across the burn matrix. Implications and applications for land managers and conservation practitioners include strategies for the promotion and maintenance of fire refugia as components of resilient forest landscapes.

Abstract Extensive high‐severity wildfires have driven major losses of ponderosa pine and mixed‐conifer forests in the southwestern United States, in some settings catalyzing enduring conversions to non‐forested vegetation types. Management interventions to reduce the probability of stand‐replacing wildfire have included mechanical fuel treatments, prescribed fire, and wildfire managed for resource benefit. In 2011, the Las Conchas fire in northern New Mexico burned forested areas not exposed to fire for >100 yr, but also reburned numerous prescribed fire units and/or areas previously burned by wildfire. At some sites, the combination of recent prescribed fire and wildfire approximated known pre‐settlement fire frequency, with two or three exposures to fire between 1977 and 2007. We analyzed gridded remotely sensed burn severity data (differenced normalized burn ratio), pre‐ and post‐fire field vegetation samples, and pre‐ and post‐fire measures of surface fuels to assess relationships and interactions between prescribed fire, prior wildfire, fuels, subsequent burn severity, and patterns of post‐fire forest retention vs. conversion to non‐forest. We found that Las Conchas burn severity was lowest, and tree survival was highest, in sites that had experienced both prescribed fire and prior wildfire. Sites that had experienced only prescribed or prior wildfire exhibited moderate burn severity and intermediate levels of forest retention. Sites lacking any recent prior fire burned at the highest severity and were overwhelmingly converted to non‐forested vegetation including grassland, oak scrub, and weedy, herbaceous‐dominated types. Burn severity in the Las Conchas fire was closely linked to surface woody fuel loads, which were reduced by prior wildfire and prescribed fire. Our results support the restoration of fire regimes via prescribed fire and resource benefit wildfire to promote the resiliency of forest types vulnerable to fire‐mediated type conversion. The application of prescribed fire to reduce surface fuels following wildfire may reduce forest loss during subsequent fire under more extreme conditions. These findings are especially relevant given likely increases in vulnerability associated with climate change impacts to wildfire and forest dynamics.

The spatial patterns resulting from large fires include refugial habitats that support surviving legacies and promote ecosystem recovery. To better understand the diverse ecological functions of refugia on burn mosaics, we used remotely sensed data to quantify neighborhood patterns of areas relatively unchanged following the 2011 Las Conchas fire. Spatial patterns of refugia measured within 10-ha moving windows varied across a gradient from areas of high density, clustered in space, to sparsely populated neighborhoods that occurred in the background matrix. The scaling of these patterns was related to the underlying structure of topography measured by slope, aspect and potential soil wetness, and spatially varying climate. Using a nonmetric multidimensional scaling analysis of species cover data collected post-Las Conchas, we found that trees and forest associates were present across the refugial gradient, but communities also exhibited a range of species compositions and potential functions. Spatial patterns of refugia quantified for three previous burns (La Mesa 1977, Dome 1996, Cerro Grande 2000) were dynamic between fire events, but most refugia persisted through at least two fires. Efforts to maintain burn heterogeneity and its ecological functions can begin with identifying where refugia are likely to occur, using terrain-based microclimate models, burn severity models and available field data.

Abstract In utero hematopoietic cell transplantation (IUHCT) utilizes fetal immune tolerance to achieve durable chimerism without conditioning or immunosuppression during a unique window in fetal development. Though donor cells have been observed within the nervous system following in utero injection, the timeline and distribution of cellular trafficking across the blood-brain barrier following IUHCT is not well understood. We injected 20 × 106 adult bone marrow mononuclear cells intravenously at gestational age (GA) 12–17 days and found that donor cells were maximally concentrated in the brain with treatment between GA 13–14. Donor cell engraftment persisted within the brain at every timepoint analyzed and concentrated within the hindbrain with significantly more grafted cells than in the forebrain. Additionally, transplanted cells terminally differentiated into various nervous system cellular morphologies and also populated the enteric nervous system. This study is the first to document the timeline and distribution of donor cell trafficking into the immune-protected nervous system and serves as a foundation for the application of IUHCT to treat neurogenetic diseases.

IntroductionChronic progressive neuroinflammation is a hallmark of neurological lysosomal storage diseases, including mucopolysaccharidosis III (MPS III or Sanfilippo disease). Since neuroinflammation is linked to white matter tract pathology, we analyzed axonal myelination and white matter density in the mouse model of MPS IIIC HgsnatP304L and post-mortem brain samples of MPS III patients.MethodsBrain and spinal cord tissues of human MPS III patients, 6-month-old HgsnatP304L mice and age- and sex-matching wild type mice were analyzed by immunofluorescence to assess levels of myelin-associated proteins, primary and secondary storage materials, and levels of microgliosis. Corpus callosum (CC) region was studied by transmission electron microscopy to analyze axon myelination and morphology of oligodendrocytes and microglia. Mouse brains were analyzed ex vivo by high-filed MRI using Diffusion Basis Spectrum Imaging in Python-Diffusion tensor imaging algorithms.ResultsAnalyses of CC and spinal cord tissues by immunohistochemistry revealed substantially reduced levels of myelin-associated proteins including Myelin Basic Protein, Myelin Associated Glycoprotein, and Myelin Oligodendrocyte Glycoprotein. Furthermore, ultrastructural analyses revealed disruption of myelin sheath organization and reduced myelin thickness in the brains of MPS IIIC mice and human MPS IIIC patients compared to healthy controls. Oligodendrocytes (OLs) in the CC of MPS IIIC mice were scarce, while examination of the remaining cells revealed numerous enlarged lysosomes containing heparan sulfate, GM3 ganglioside or “zebra bodies” consistent with accumulation of lipids and myelin fragments. In addition, OLs contained swollen mitochondria with largely dissolved cristae, resembling those previously identified in the dysfunctional neurons of MPS IIIC mice. Ex vivo Diffusion Basis Spectrum Imaging revealed compelling signs of demyelination (26% increase in radial diffusivity) and tissue loss (76% increase in hindered diffusivity) in CC of MPS IIIC mice.DiscussionOur findings demonstrate an important role for white matter injury in the pathophysiology of MPS III. This study also defines specific parameters and brain regions for MRI analysis and suggests that it may become a crucial non-invasive method to evaluate disease progression and therapeutic response.

Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB) is a recessive genetic disorder that severely affects the brain due to a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intra-lysosomal accumulation of partially degraded heparan sulfate. There are no effective treatments for this disorder. In this project, we carried out an ex vivo correction of neural stem cells derived from Naglu−/− mice (iNSCs) induced pluripotent stem cells (iPSC) using a modified enzyme in which human NAGLU is fused to an insulin-like growth factor II receptor binding peptide in order to improve enzyme uptake. After brain transplantation of corrected iNSCs into Naglu−/− mice and long-term evaluation of their impact, we successfully detected NAGLU-IGFII activity in all transplanted animals. We found decreased lysosomal accumulation and reduced astrocytosis and microglial activation throughout transplanted brains. We also identified a novel neuropathological phenotype in untreated Naglu−/− brains with decreased levels of the neuronal marker Map2 and accumulation of synaptophysin-positive aggregates. Upon transplantation, we restored levels of Map2 expression and significantly reduced formation of synaptophysin-positive aggregates. Our findings suggest that genetically engineered iNSCs can be used to effectively deliver the missing enzyme to the brain and treat Sanfilippo type B-associated neuropathology.

Although a disease-modifying therapy for classic late infantile neuronal ceroid lipofuscinosis (CLN2 disease) exists, poor understanding of cellular pathophysiology has hampered the development of more effective and persistent therapies. Here, we investigated the nature and progression of neurological and underlying neuropathological changes in Cln2R207X mice, which carry one of the most common pathogenic mutations in human patients but are yet to be fully characterized. Long-term electroencephalography recordings revealed progressive epileptiform abnormalities, including spontaneous seizures, providing a robust, quantifiable, and clinically relevant phenotype. These seizures were accompanied by the loss of multiple cortical neuron populations, including those stained for interneuron markers. Further histological analysis revealed early localized microglial activation months before neuron loss started in the thalamocortical system and spinal cord, which was accompanied by astrogliosis. This pathology was more pronounced and occurred in the cortex before the thalamus or spinal cord and differed markedly from the staging seen in mouse models of other forms of neuronal ceroid lipofuscinosis. Neonatal administration of adeno-associated virus serotype 9–mediated gene therapy ameliorated the seizure and gait phenotypes and prolonged the life span of Cln2R207X mice, attenuating most pathological changes. Our findings highlight the importance of clinically relevant outcome measures for judging preclinical efficacy of therapeutic interventions for CLN2 disease.

Abstract Cannabidiol (CBD) has gained attention as a therapeutic agent and is purported to have immunomodulatory, neuroprotective, and anti-seizure effects. Here, we determined the effects of chronic CBD administration in a mouse model of CLN1 disease (Cln1 −/−) that simultaneously exhibits neuroinflammation, neurodegeneration, and spontaneous seizures. Proteomic analysis showed that putative CBD receptors are expressed at similar levels in the brains of Cln1 −/− mice compared to normal animals. Cln1 −/− mice received an oral dose (100 mg/kg/day) of CBD for six months and were evaluated for changes in pathological markers of disease and seizures. Chronic cannabidiol administration was well-tolerated, high levels of CBD were detected in the brain, and markers of astrocytosis and microgliosis were reduced. However, CBD had no apparent effect on seizure frequency or neuron survival. These data are consistent with CBD having immunomodulatory effects. It is possible that a higher dose of CBD could also reduce neurodegeneration and seizure frequency.

BackgroundThe Neuronal Ceroid Lipofuscinoses (NCLs) may be considered distinct neurodegenerative disorders with separate underlying molecular causes resulting from monogenetic mutations. An alternative hypothesis is to consider the NCLs as related diseases that share lipofuscin pathobiology as the common core feature, but otherwise distinguished by different a) initial anatomic location, and b) disease propagation.MethodsWe have tested this hypothesis by comparing known differences in symptomatology and pathology of the CLN1 phenotype caused by complete loss of PPT1 function (i.e., the classical infantile form) and of the classical juvenile CLN3 phenotype. These two forms of NCL represent early onset and rapidly progressing vs. late onset and slowly progressing disease modalities respectively.ResultsDespite displaying similar pathological endpoints, the clinical phenotypes and the evidence of imaging and postmortem studies reveal strikingly different time courses and distributions of disease propagation. Data from CLN1 disease are indicative of disease propagation from the body, with early effects within the spinal cord and subsequently within the brainstem, the cerebral hemispheres, cerebellum and retina. In contrast, the retina appears to be the most vulnerable organ in CLN3, and the site where pathology is first present. Pathology subsequently is present in the occipital connectome of the CLN3 brain, followed by a top-down propagation in which cerebral and cerebellar atrophy in early adolescence is followed by involvement of the peripheral nerves in later adolescence/early twenties, with the extrapyramidal system also affected during this time course.DiscussionThe propagation of disease in these two NCLs therefore has much in common with the “Brain-first” vs. “Body-first” models of alpha-synuclein propagation in Parkinson's disease. CLN1 disease represents a “Body-first” or bottom-up disease propagation and CLN3 disease having a “Brain-first” and top-down propagation. It is noteworthy that the varied phenotypes of CLN1 disease, whether it starts in infancy (infantile form) or later in childhood (juvenile form), still fit with our proposed hypothesis of a bottom-up disease propagation in CLN1. Likewise, in protracted CLN3 disease, where both cognitive and motor declines are delayed, the initial manifestations of disease are also seen in the outer retinal layers, i.e., identical to classical Juvenile NCL disease.

CLN1 disease, also called infantile neuronal ceroid lipofuscinosis (NCL) or infantile Batten disease, is a fatal neurodegenerative lysosomal storage disorder resulting from mutations in the CLN1 gene encoding the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1). Therapies for CLN1 disease have proven challenging because of the aggressive disease course and the need to treat widespread areas of the brain and spinal cord. Indeed, gene therapy has proven less effective for CLN1 disease than for other similar lysosomal enzyme deficiencies. We therefore tested the efficacy of enzyme replacement therapy (ERT) by administering monthly infusions of recombinant human PPT1 (rhPPT1) to PPT1-deficient mice (Cln1–/–) and CLN1R151X sheep to assess how to potentially scale up for translation. In Cln1–/– mice, intracerebrovascular (i.c.v.) rhPPT1 delivery was the most effective route of administration, resulting in therapeutically relevant CNS levels of PPT1 activity. rhPPT1-treated mice had improved motor function, reduced disease-associated pathology, and diminished neuronal loss. In CLN1R151X sheep, i.c.v. infusions resulted in widespread rhPPT1 distribution and positive treatment effects measured by quantitative structural MRI and neuropathology. This study demonstrates the feasibility and therapeutic efficacy of i.c.v. rhPPT1 ERT. These findings represent a key step toward clinical testing of ERT in children with CLN1 disease and highlight the importance of a cross-species approach to developing a successful treatment strategy.

Batten disease is a family of rare, fatal, neuropediatric diseases presenting with memory/learning decline, blindness, and loss of motor function. Recently, we reported the use of an AAV9-mediated gene therapy that prevents disease progression in a mouse model of CLN6-Batten disease (Cln6nclf), restoring lifespans in treated animals. Despite the success of our viral-mediated gene therapy, the dosing strategy was optimized for delivery to the brain parenchyma and may limit the therapeutic potential to other disease-relevant tissues, such as the eye. Here, we examine whether cerebrospinal fluid (CSF) delivery of scAAV9.CB.CLN6 is sufficient to ameliorate visual deficits in Cln6nclf mice. We show that intracerebroventricular (i.c.v.) delivery of scAAV9.CB.CLN6 completely prevents hallmark Batten disease pathology in the visual processing centers of the brain, preserving neurons of the superior colliculus, thalamus, and cerebral cortex. Importantly, i.c.v.-delivered scAAV9.CB.CLN6 also expresses in many cells throughout the central retina, preserving many photoreceptors typically lost in Cln6nclf mice. Lastly, scAAV9.CB.CLN6 treatment partially preserved visual acuity in Cln6nclf mice as measured by optokinetic response. Taken together, we report the first instance of CSF-delivered viral gene reaching and rescuing pathology in both the brain parenchyma and retinal neurons, thereby partially slowing visual deterioration.

Abstract Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24–30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

While significant efforts have been made in developing pre-clinical treatments for the neuronal ceroid lipofuscinoses (NCLs), many challenges still remain to bring children with NCLs a cure. Devising effective therapeutic strategies for the NCLs will require a better understanding of pathophysiology, but little is known about the mechanisms by which loss of lysosomal proteins causes such devastating neurodegeneration. Research into glial cells including astrocytes, microglia, and oligodendrocytes have revealed many of their critical functions in brain homeostasis and potential contributions to neurodegenerative diseases. Genetically modified mouse models have served as a useful platform to define the disease progression in the central nervous system across NCL subtypes, revealing a wide range of glial responses to disease. The emerging evidence of glial dysfunction questions the traditional “neuron-centric” view of NCLs, and would suggest that directly targeting glia in addition to neurons could lead to better therapeutic outcomes. This review summarizes the most up-to-date understanding of glial pathologies and their contribution to the pathogenesis of NCLs, and highlights some of the associated challenges that require further research.

Issue: The impact of neurological disorders is recognised globally, with one in six people affected in their lifetime and few treatments to slow or halt disease progression. This is due in part to the increasing ageing population, and is confounded by the high failure rate of translation from rodent-derived therapeutics to clinically effective human neurological interventions. Improved translation is demonstrated using higher order mammals with more complex/comparable neuroanatomy. These animals effectually span this translational disparity and increase confidence in factors including routes of administration/dosing and ability to scale, such that potential therapeutics will have successful outcomes when moving to patients. Coupled with advancements in genetic engineering to produce genetically tailored models, livestock are increasingly being used to bridge this translational gap. Approach: In order to aid in standardising characterisation of such models, we provide comprehensive neurological assessment protocols designed to inform on neuroanatomical dysfunction and/or lesion(s) for large animal species. We also describe the applicability of these exams in different large animals to help provide a better understanding of the practicalities of cross species neurological disease modelling. Recommendation: We would encourage the use of these assessments as a reference framework to help standardise neurological clinical scoring of large animal models.

Sanfilippo syndrome type B (mucopolysaccharidosis type IIIB [MPS IIIB]) is a lysosomal storage disorder primarily affecting the brain that is caused by a deficiency in the enzyme α-N-acetylglucosaminidase (NAGLU), leading to intralysosomal accumulation of heparan sulfate. There are currently no treatments for this disorder. Here we report that, ex vivo, lentiviral correction of Naglu−/− neural stem cells derived from Naglu−/− mice (iNSCs) corrected their lysosomal pathology and allowed them to secrete a functional NAGLU enzyme that could be taken up by deficient cells. Following long-term transplantation of these corrected iNSCs into Naglu−/− mice, we detected NAGLU activity in the majority of engrafted animals. Successfully transplanted Naglu−/− mice showed a significant decrease in storage material, a reduction in astrocyte activation, and complete prevention of microglial activation within the area of engrafted cells and neighboring regions, with beneficial effects extending partway along the rostrocaudal axis of the brain. Our results demonstrate long-term engraftment of iNSCs in the brain that are capable of cross-correcting pathology in Naglu−/− mice. Our findings suggest that genetically engineered iNSCs could potentially be used to deliver enzymes and treat MPS IIIB. Keywords: MPS IIIB, lysosomal storage disorder, stem cell therapy

Abstract The neuronal ceroid lipofuscinoses (NCLs) are the most common cause of childhood dementia and are invariably fatal. Early localized glial activation occurs in these disorders, and accurately predicts where neuronal loss is most pronounced. Recent evidence suggests that glial dysfunction may contribute to neuron loss, and we have now explored this possibility in infantile NCL (INCL, CLN1 disease). We grew primary cultures of astrocytes, microglia, and neurons derived from Ppt1 deficient mice (Ppt1 −/− ) and assessed their properties compared to wildtype (WT) cultures, before co-culturing them in different combinations (astrocytes with microglia, astrocytes or microglia with neurons, all three cell types together). These studies revealed that both Ppt1 −/− astrocytes and microglia exhibit a more activated phenotype under basal unstimulated conditions, as well as alterations to their protein expression profile following pharmacological stimulation. Ppt1 - /− astrocytes also displayed abnormal calcium signalling and an elevated cytoplasmic Ca2+ level, and a profound defect in their survival. Ppt1 −/− neurons displayed decreased neurite outgrowth, altered complexity, a reduction in cell body size, and impaired neuron survival with prolonged time in culture. In co-cultures, the presence of both astrocytes and microglia from Ppt1 −/− mice further impaired the morphology of both wild type and Ppt1 −/− neurons. This negative influence was more pronounced for Ppt1 −/− microglia, which appeared to trigger increased Ppt1 −/− neuronal death. In contrast, wild type glial cells, especially astrocytes, ameliorated some of the morphological defects observed in Ppt1 −/− neurons. These findings suggest that both Ppt1 −/− microglia and astrocytes are dysfunctional and may contribute to the neurodegeneration observed in CLN1 disease. However, the dysfunctional phenotypes of Ppt1 −/− glia are different from those present in CLN3 disease, suggesting that the pathogenic role of glia may differ between NCLs.

Antibodies against recombinant proteins can significantly reduce their effectiveness in unanticipated ways. We evaluated the humoral response of mice with the lysosomal storage disease mucopolysaccharidosis type I treated with weekly intravenous recombinant human alpha-l-iduronidase (rhIDU). Unlike patients, the majority of whom develop antibodies to recombinant human alpha-l-iduronidase, only approximately half of the treated mice developed antibodies against recombinant human alpha-l-iduronidase and levels were low. Serum from antibody-positive mice inhibited uptake of recombinant human alpha-l-iduronidase into human fibroblasts by partial inhibition compared to control serum. Tissue and cellular distributions of rhIDU were altered in antibody-positive mice compared to either antibody-negative or naive mice, with significantly less recombinant human alpha-l-iduronidase activity in the heart and kidney in antibody-positive mice. In the liver, recombinant human alpha-l-iduronidase was preferentially found in sinusoidal cells rather than in hepatocytes in antibody-positive mice. Antibodies against recombinant human alpha-l-iduronidase enhanced uptake of recombinant human alpha-l-iduronidase into macrophages obtained from MPS I mice. Collectively, these results imply that a humoral immune response against a therapeutic protein can shift its distribution preferentially into macrophage-lineage cells, causing decreased availability of the protein to the cells that are its therapeutic targets.

Abstract 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.

The transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis. Here authors show that trehalose, an mTOR-independent autophagy inducer, alleviates the pathological phenotypes in a mouse model of neurodegenerative disease. Trehalose acts by inhibiting Akt, which normally suppresses TFEB via an mTORC1-independent mechanism.

Batten disease, or juvenile NCL, is a fatal neurodegenerative disorder that occurs due to mutations in the CLN3 gene. Because the function of CLN3 remains unclear, experimental therapies for JNCL have largely concentrated upon the targeting of downstream pathomechanisms. Neuron loss is preceded by localized glial activation, and in this proof-of-concept study we have investigated whether targeting this innate immune response with ibuprofen in combination with the neuroprotective agent lamotrigine improves the previously documented beneficial effects of immunosuppressants alone. Drugs were administered daily to symptomatic Cln3−/− mice over a 3 month period, starting at 6 months of age, and their impact was assessed using both behavioral and neuropathological outcome measures. During the treatment period, the combination of ibuprofen and lamotrigine significantly improved the performance of Cln3−/− mice on the vertical pole test, slowing the disease-associated decline, but had less of an impact upon their rotarod performance. There were also moderate and regionally dependent effects upon astrocyte activation that were most pronounced for ibuprofen alone, but there was no overt effect upon microglial activation. Administering such treatments for longer periods will enable testing for any impact upon the neuron loss that occurs later in disease progression. Given the partial efficacy of these treatments, it will be important to test further drugs of this type in order to find more effective combinations.

Nature Communications 8: Article number: 14338 (2017); Published: 6 February 2017; Updated: 13 June 2017 This Article contains errors in Figs 2 and 3, for which we apologize. In Fig. 2c, the four images were inadvertently duplicated from the images in Fig. 2b. In Fig. 3g, the image at the upper right corner, corresponding to the condition UT_ Cln3Δex7-8 was inadvertently duplicated from the image in the lower right corner of Fig.

SUMMARY Both CLN1 and CLN5 deficiencies lead to severe neurodegenerative diseases of childhood, known as neuronal ceroid lipofuscinoses (NCLs). The broadly similar phenotypes of NCL mouse models, and the potential for interactions between NCL proteins, raise the possibility of shared or convergent disease mechanisms. To begin addressing these issues, we have developed a new mouse model lacking both Cln1 and Cln5 genes. These double-knockout (Cln1/5 dko) mice were fertile, showing a slight decrease in expected Mendelian breeding ratios, as well as impaired embryoid body formation by induced pluripotent stem cells derived from Cln1/5 dko fibroblasts. Typical disease manifestations of the NCLs, i.e. seizures and motor dysfunction, were detected at the age of 3 months, earlier than in either single knockout mouse. Pathological analyses revealed a similar exacerbation and earlier onset of disease in Cln1/5 dko mice, which exhibited a pronounced accumulation of autofluorescent storage material. Cortical demyelination and more pronounced glial activation in cortical and thalamic regions was followed by cortical neuron loss. Alterations in lipid metabolism in Cln1/5 dko showed a specific increase in plasma phospholipid transfer protein (PLTP) activity. Finally, gene expression profiling of Cln1/5 dko cortex revealed defects in myelination and immune response pathways, with a prominent downregulation of α-synuclein in Cln1/5 dko mouse brains. The simultaneous loss of both Cln1 and Cln5 genes might enhance the typical pathological phenotypes of these mice by disrupting or downregulating shared or convergent pathogenic pathways, which could potentially include interactions of CLN1 and CLN5.

The neuronal ceroid lipofuscinoses constitute the most common group of childhood neurodegenerative disorders. These devastating disorders still remain without effective treatment. The use of animal models has provided significant information about NCL pathogenesis, highlighting early glial activation and neuron loss in specific brain regions of affected animals. Here, we have characterized the timing and regional-specificity of the pathological events of CLN8 disease utilizing the Cln8 deficient mouse model, Cln8mnd. We have studied the progression of neuron loss, astrocytosis and microglial activation from early to moderately symptomatic (1, 3 and 5 months) and late symptomatic (8 months) mice. In Cln8 deficiency, the somatosensory pathway comprising the thalamic ventral posterior nucleus (VPM/VPL) and the primary somatosensory cortex (S1BF) was found to be the most affected relay system. Scattered microglia that appeared partially activated were already present at 3 months of age, followed by astrocytosis and the loss of thalamic relay neurons at 5 months of age, with all these phenotypes and glial activation becoming more pronounced with disease progression. Reactive changes followed a similar pattern in the corresponding cortical target regions, but only moderate neuron loss was detected. Compared to the somatosensory system, in the visual thalamocortical pathway, neuron loss appeared relatively late in the disease, at 8 months. Neuron loss was preceded by glial activation in the dorsal lateral geniculate nucleus (LGNd) and in the primary visual cortex (V1). Taken together these data highlight the pathological targeting of the somatosensory thalamocortical pathway in Cln8 deficiency, in common with other forms of NCL. However, in contrast to other previously characterized NCL models, the Cln8mnd mouse shows relatively mild and late appearing pathology within the thalamocortical visual pathway.