Axonal Spheroids Research Articles

Pathologic basis of the preferential thinning of thecorpus callosum in adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP).

BackgroundAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is an early onset dementia characterized by axonal loss in the cerebral white matter with swollen axons (spheroids). It had been reported that the preferential thinning and “focal lesions” of the corpus callosum were observed on T2-weighted MRI in ALSP patients. The present study aimed to reveal the pathologic basis of them in relation to brain lesion staging (I ~ IV: Oyanagi et al. 2017).MethodsSeven autopsied brains of ALSP and five controls were neuropathologically examined.ResultsEven at Stage I, corpus callosum body showed evident atrophy, and the atrophy advanced with stage progression. Spheroid size and density were maximal at Stage II in both centrum semiovale and corpus callosum body, but spheroids were larger in corpus callosum body than in centrum semiovale. Microglia in the body at Stage II had a larger cytoplasm than those in centrum semiovale. But spheroids and microglia in the “focal lesions” were identical with those of centrum semiovale.ConclusionPreferential thinning of corpus callosum was considered to be formed in relation to peculiar morphological alteration of microglia there in ALSP. Instead, “focal lesions” were formed in connection with the lesions in centrum semiovale.

Modeling Brain Pathology of Niemann‐Pick Disease Type C Using Patient‐Derived Neurons

AbstractBackgroundNiemann‐Pick disease type C (NPC) is a rare autosomal‐recessive lysosomal storage disease that is also associated with progressive neurodegeneration. NPC shares many pathological features with Alzheimer's disease, including neurofibrillary tangles, axonal spheroids, β‐amyloid deposition, and dystrophic neurites. Here, we examined if these pathological features could be detected in induced pluripotent stem cell (iPSC)‐derived neurons from NPC patients.MethodsBrain tissues from 8 NPC patients and 5 controls were analyzed for histopathological and biochemical markers of pathology. To model disease in culture, iPSCs from NPC patients and controls were differentiated into cortical neurons.ResultsWe found hyperphosphorylated tau, altered processing of amyloid precursor protein, and increased Aβ42 in NPC postmortem brains and in iPSC‐derived cortical neurons from NPC patients.ConclusionOur findings demonstrated that the main pathogenic phenotypes typically found in NPC brains were also observed in patient‐derived neurons, providing a useful model for further mechanistic and therapeutic studies of NPC. © 2021 International Parkinson and Movement Disorder Society

Loss of homeostatic microglia in rare neurological disorders: implications for cell transplantation

Microglia originating from yolk sac exert various functions to maintain the homeostasis in the brain, and their functional breakdown appears to be involved in the pathophysiology of various neurological diseases. In this review article, loss of homeostatic microglia and new therapeutic approaches for rare neurological disorders are discussed. ASLP (adult-onset leukoencephalopathy with axonal spheroids and pigmented glia) known as a primary microgliopathy is an adult-onset leukoencephalopathy caused by CSF1R mutation. CSF1 receptor encoded by CSF1R plays an important role in the function of microglia. In brain of ALSP patients, homeostatic microglia are significantly reduced. The biallelic mutations for CSF1R cause childhood-onset severe phenotype and elimination of microglia from the brain parenchyma. Since microglia also almost disappear in CSF1R-deficient mice and rats, CSF1R deficiency and loss of microglia appear to be tightly associated across species. Based on the underlying mechanism of homeostatic microglia loss, novel approaches using cell transplantation of normal microglia-like cells have been attempted. Transplantation of wild-type bone marrow cells into Csf1r-/- mice results in replacement by donor-derived microglial-like cells in the recipient's brain. The concept of "microglial niche" may explain the rationale behind the microglial cell transplantation in disease condition(s). Hematopoietic stem cell transplantation (HSCT) has been attempted in 4 patients with ALSP. Beneficial effects by showing stabilization of the disease course have been observed. Although the effectiveness of HSCT for ALSP patients warrants further investigation, the approach of cell transplantation that replaces ruptured homeostatic microglia with normal microglia-like cells seems to be promising.

Attenuated CSF‐1R signalling drives cerebrovascular pathology

Cerebrovascular pathologies occur in up to 80% of cases of Alzheimer's disease; however, the underlying mechanisms that lead to perivascular pathology and accompanying blood–brain barrier (BBB) disruption are still not fully understood. We have identified previously unreported mutations in colony stimulating factor‐1 receptor (CSF‐1R) in an ultra‐rare autosomal dominant condition termed adult‐onset leucoencephalopathy with axonal spheroids and pigmented glia (ALSP). Cerebrovascular pathologies such as cerebral amyloid angiopathy (CAA) and perivascular p‐Tau were some of the primary neuropathological features of this condition. We have identified two families with different dominant acting alleles with variants located in the kinase region of the CSF‐1R gene, which confer a lack of kinase activity and signalling. The protein product of this gene acts as the receptor for 2 cognate ligands, namely colony stimulating factor‐1 (CSF‐1) and interleukin‐34 (IL‐34). Here, we show that depletion in CSF‐1R signalling induces BBB disruption and decreases the phagocytic capacity of peripheral macrophages but not microglia. CSF‐1R signalling appears to be critical for macrophage and microglial activation, and macrophage localisation to amyloid appears reduced following the induction of Csf‐1r heterozygosity in macrophages. Finally, we show that endothelial/microglial crosstalk and concomitant attenuation of CSF‐1R signalling causes re‐modelling of BBB‐associated tight junctions and suggest that regulating BBB integrity and systemic macrophage recruitment to the brain may be therapeutically relevant in ALSP and other Alzheimer’s‐like dementias.

Compound heterozygous PLA2G6 loss-of-function variants in Swaledale sheep with neuroaxonal dystrophy

Sporadic occurrences of neurodegenerative disorders including neuroaxonal dystrophy (NAD) have been previously reported in sheep. However, so far no causative genetic variant has been found for ovine NAD. The aim of this study was to characterize the phenotype and the genetic aetiology of an early-onset neurodegenerative disorder observed in several lambs of purebred Swaledale sheep, a native English breed. Affected lambs showed progressive ataxia and stiff gait and subsequent histopathological analysis revealed the widespread presence of axonal spheroid indicating neuronal degeneration. Thus, the observed clinical phenotype could be explained by a novel form of NAD. After SNP genotyping and subsequent linkage mapping within a paternal half-sib pedigree with a total of five NAD-affected lambs, we identified two loss-of-function variants by whole-genome sequencing in the ovine PLA2G6 gene situated in a NAD-linked genome region on chromosome 3. All cases were carriers of a compound heterozygous splice site variant in intron 2 and a nonsense variant in exon 8. Herein we present evidence for the occurrence of a familial novel form of recessively inherited NAD in sheep due to allelic heterogeneity at PLA2G6. This study reports two pathogenic variants in PLA2G6 causing a novel form of NAD in Swaledale sheep which enables selection against this fatal disorder.

Amyloid-β PET and CSF in an autopsy-confirmed cohort.

ObjectiveAccumulation of amyloid‐β is among the earliest changes in Alzheimer’s disease (AD). Amyloid‐β positron emission tomography (PET) and Aβ 42 in cerebrospinal fluid (CSF) both assess amyloid‐β pathology in‐vivo, but 10–20% of cases show discordant (CSF+/PET− or CSF‐/PET+) results. The neuropathological correspondence with amyloid‐β CSF/PET discordance is unknown.MethodsWe included 21 patients from our tertiary memory clinic who had undergone both CSF Aβ 42 analysis and amyloid‐β PET, and had neuropathological data available. Amyloid‐β PET and CSF results were compared with neuropathological ABC scores (comprising of Thal (A), Braak (B), and CERAD (C) stage, all ranging from 0 [low] to 3 [high]) and neuropathological diagnosis.ResultsNeuropathological diagnosis was AD in 11 (52%) patients. Amyloid‐β PET was positive in all A3, C2, and C3 cases and in one of the two A2 cases. CSF Aβ 42 was positive in 92% of ≥A2 and 90% of ≥C2 cases. PET and CSF were discordant in three of 21 (14%) cases: CSF+/PET− in a patient with granulomatosis with polyangiitis (A0B0C0), CSF+/PET− in a patient with FTLD‐TDP type B (A2B1C1), and CSF‐/PET+ in a patient with AD (A3B3C3). Two CSF+/PET+ cases had a non‐AD neuropathological diagnosis, that is FTLD‐TDP type E (A3B1C1) and adult‐onset leukoencephalopathy with axonal spheroids (A1B1C0).InterpretationOur study demonstrates neuropathological underpinnings of amyloid‐β CSF/PET discordance. Furthermore, amyloid‐β biomarker positivity on both PET and CSF did not invariably result in an AD diagnosis at autopsy, illustrating the importance of considering relevant comorbidities when evaluating amyloid‐β biomarker results.

Microglial reduction of colony stimulating factor-1 receptor expression is sufficient to confer adult onset leukodystrophy.

Adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a dementia resulting from dominantly inherited CSF1R inactivating mutations. The Csf1r+/- mouse mimics ALSP symptoms and pathology. Csf1r is mainly expressed in microglia, but also in cortical layer V neurons that are gradually lost in Csf1r+/- mice with age. We therefore examined whether microglial or neuronal Csf1r loss caused neurodegeneration in Csf1r+/- mice. The behavioral deficits, pathologies and elevation of Csf2 expression contributing to disease, previously described in the Csf1r+/- ALSP mouse, were reproduced by microglial deletion (MCsf1rhet mice), but not by neural deletion. Furthermore, increased Csf2 expression by callosal astrocytes, oligodendrocytes, and microglia was observed in Csf1r+/- mice and, in MCsf1rhet mice, the densities of these three cell types were increased in supraventricular patches displaying activated microglia, an early site of disease pathology. These data confirm that ALSP is a primary microgliopathy and inform future therapeutic and experimental approaches.

Advancing treatment for CSF1R-related leukoencephalopathy

Objective: Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a neurodegenerative disease characterized by the rapid progression of neurologic symptoms including frontotemporal dementia, pyramidal signs, and parkinsonism. The disease is associated with mutations of the colony stimulating factor 1 receptor (CSF1R) gene, which is critical for proper microglial function. Treatments targeting microglial restoration are now being investigated. We hypothesize that haematopoietic stem cell transplantation (HSCT) is an effective treatment for ALSP by replenishing a healthy microglial population.

IP3R-mediated intra-axonal Ca2+ release contributes to secondary axonal degeneration following contusive spinal cord injury

Secondary axonal loss contributes to the persistent functional disability following trauma. Consequently, preserving axons following spinal cord injury (SCI) is a major therapeutic goal to improve neurological outcome; however, the complex molecular mechanisms that mediate secondary axonal degeneration remain unclear. We previously showed that IP3R-mediated Ca2+ release contributes to axonal dieback and axonal loss following an ex vivo laser-induced SCI. Nevertheless, targeting IP3R in a clinically relevant in vivo model of SCI and determining its contribution to secondary axonal degeneration has yet to be explored. Here we used intravital two-photon excitation microscopy to assess the role of IP3R in secondary axonal degeneration in real-time after a contusive-SCI in vivo. To visualize Ca2+ changes specifically in spinal axons over time, adult 6–8 week-old triple transgenic Avil-Cre:Ai9:Ai95 (sensory neuron-specific expression of tdTomato and the genetic calcium indicator GCaMP6f) mice were subjected to a mild (30 kdyn) T12 contusive-SCI and received delayed treatment with the IP3R blocker 2-APB (100 μM, intrathecal delivery at 3, and 24 h following injury) or vehicle control. To determine the IP3R subtype involved, we knocked-down IP3R3 using capped phosphodiester oligonucleotides. Delayed treatment with 2-APB significantly reduced axonal spheroids, increased axonal survival, and reduced intra-axonal Ca2+ accumulation within dorsal column axons at 24 h following SCI in vivo. Additionally, knockdown of IP3R3 yielded increased axon survival 24 h post-SCI. These results suggest that IP3R-mediated Ca2+ release contributes to secondary axonal degeneration in vivo following SCI.

Regulation of degenerative spheroids after injury

Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1−/− and DR6−/−, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1−/− and DR6−/−, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

Cleavage of human tau at Asp421 inhibits hyperphosphorylated tau induced pathology in a Drosophila model

Hyperphosphorylated and truncated tau variants are enriched in neuropathological aggregates in diseases known as tauopathies. However, whether the interaction of these posttranslational modifications affects tau toxicity as a whole remains unresolved. By expressing human tau with disease-related Ser/Thr residues to simulate hyperphosphorylation, we show that despite severe neurodegeneration in full-length tau, with the truncation at Asp421, the toxicity is ameliorated. Cytological and biochemical analyses reveal that hyperphosphorylated full-length tau distributes in the soma, the axon, and the axonal terminal without evident distinction, whereas the Asp421-truncated version is mostly restricted from the axonal terminal. This discrepancy is correlated with the fact that fly expressing hyperphosphorylated full-length tau, but not Asp421-cleaved one, develops axonopathy lesions, including axonal spheroids and aberrant actin accumulations. The reduced presence of hyperphosphorylated tau in the axonal terminal is corroborated with the observation that flies expressing Asp421-truncated variants showed less motor deficit, suggesting synaptic function is preserved. The Asp421 cleavage of tau is a proteolytic product commonly found in the neurofibrillary tangles. Our finding suggests the coordination of different posttranslational modifications on tau may have an unexpected impact on the protein subcellular localization and cytotoxicity, which may be valuable when considering tau for therapeutic purposes.

Altered features of monocytes in adult onset leukoencephalopathy with axonal spheroids and pigmented glia: A clue to the pathomechanism of microglial dyshomeostasis

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is an autosomal-dominant type of leukoencephalopathy caused by gene mutation of colony stimulating factor 1 receptor, which is expressed mainly on monocyte lineage cells such as monocytes in the peripheral blood and microglia in the brain. Hence, microglial dysfunction is regarded as critical in the pathogenesis of ALSP. However, functional changes in these cells have not been elucidated. In this study, we report the phenotypic and functional alterations of monocytes in four patients with ALSP. Flow cytometric analysis revealed altered expression of antigen presentation- and migration-related molecules, an inflammatory shift in cytokine production and phagocytic impairment in ALSP monocytes. We speculate that the observed altered features of monocytes are mostly shared by microglial cells, leading to the clinical history and pathological characteristics of ALSP. Our analysis of PB monocytes provides novel insights into the pathogenesis of ALSP.

Intoxication by Astragalus garbancillo var. garbancillo in llamas.

Lysosomal storage diseases are inherited and acquired disorders characterized by dysfunctional lysosomes. Intracytoplasmic accumulation of undegraded substrates leads to impaired cellular function and death. Several plant species are toxic to livestock because of the presence of indolizidine alkaloids, including swainsonine, which cause a storage disease. Swainsonine-induced nervous disease (i.e., locoism) of sheep and cattle is well recognized in several parts of the world, particularly in the western United States and in parts of Australia. Spontaneous intoxication by Astragalus garbancillo var. garbancillo was suspected in a group of 70 llamas (Lama glama) in Jujuy Province, northwestern Argentina. The animals grazed an area dominated by stands of A. garbancillo var. garbancillo. Clinical signs were staggering, ataxia, hypermetria, and progressive weight loss. The clinical course in individual animals was ~50 d. The main microscopic changes were Purkinje cell degeneration, necrosis, and loss, associated with intracytoplasmic vacuolation, meganeurite formation, and Wallerian degeneration. Specific positive labeling for ubiquitin was observed in axonal spheroids. Composite leaf and stem samples of A. garbancillo var. garbancillo analyzed by high-performance liquid chromatography contained 0.03% swainsonine. Based on the microscopic lesions, clinical history, and plant analysis, a diagnosis was made of storage disease caused by consumption of swainsonine-containing A. garbancillo var. garbancillo.

Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role ofelevated cerebral Csf2 expression. Here, we showthat monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions.

Repeat intravital imaging of the murine spinal cord reveals degenerative and reparative responses of spinal axons in real-time following a contusive SCI

Spinal cord injury (SCI) induces a secondary degenerative response that causes the loss of spared axons and worsens neurological outcome. The complex molecular mechanisms that mediate secondary axonal degeneration remain poorly understood. To further our understanding of secondary axonal degeneration following SCI, we assessed the spatiotemporal dynamics of axonal spheroid and terminal bulb formation following a contusive SCI in real-time in vivo. Adult 6–8 week old Thy1YFP transgenic mice underwent a T12 laminectomy for acute imaging sessions or were implanted with a custom spinal cord imaging chamber for chronic imaging of the spinal cord. Two-photon excitation time-lapse microscopy was performed prior to a mild contusion SCI (30 kilodyne, IH Impactor) and at 1–4 h and 1–14 days post-SCI. We quantified the number of axonal spheroids, their size and distribution, the number of endbulbs, and axonal survival from 1 h to 14 days post-SCI. Our data reveal that the majority of axons underwent swelling and axonal spheroid formation acutely after SCI resulting in the loss of ~70% of axons by 1 day after injury. In agreement, the number of axonal spheroids rapidly increased at 1 h after SCI and remained significantly elevated up to 14 days after SCI. Furthermore, the distribution of axonal spheroids spread mediolaterally over time indicative of delayed secondary degenerative processes. In contrast, axonal endbulbs were relatively sparse and their numbers peaked at 1 day after injury. Intriguingly, axonal survival significantly increased at 7 and 14 days compared to 3 days after SCI revealing a potential endogenous axonal repair process that mirrors the known spontaneous functional recovery after SCI. In support, ~43% of tracked axonal spheroids resolved over the course of observation revealing their dynamic nature. Furthermore, axonal spheroids and endbulbs accumulated mitochondria and excessive tubulin polyglutamylation suggestive of disrupted axonal transport as a shared mechanism. Collectively, this study provides important insight into both degenerative and recoverable responses of axons following contusive SCI in real-time. Understanding how axons spontaneously recover after SCI will be an important avenue for future SCI research and may help guide future clinical trials.

Sporadic Japanese case of adult‐onset leukoencephalopathy with axonal spheroids and pigmented glia caused by a de novo p.Phe849del mutation in CSF1R

AbstractAdult‐onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is an autosomal dominant neurodegenerative disorder caused by mutations in the colony‐stimulating factor 1 receptor (CSF1R) gene. We, herein, report the first Japanese case of ALSP caused by a de novo p.Phe849del mutation in CSF1R: a 44‐year‐old man who presented callosal disconnection symptoms. Brain MRI revealed dilation of the lateral ventricles, periventricular white matter hyperintensities, and thinning of the corpus callosum with hyperintensities on T2‐weighted and FLAIR images. Brain single photon emission computed tomography (SPECT) showed hypoperfusion in the anterior corpus callosum. White matter lesions in MRI and hypoperfusion in SPECT increased with age. A brain biopsy at 44 years revealed axonal spheroids. Callosal disconnection symptoms and hypoperfusion in the anterior corpus callosum may be important indicators of ALSP, especially when caused by a p.Phe849del CSF1R mutation.

Loss of Ramified Microglia Precedes Axonal Spheroid Formation in Adult-Onset Leukoencephalopathy with Axonal Spheroids.

Two different pathological mechanisms have been suggested to underlie adult-onset leukoencephalopathy with axonal spheroids (ALAS). Pathological studies have suggested that ALAS involves primary axonopathy with secondary demyelination. However, the identification of mutations in Colony Stimulating Factor 1 Receptor (CSF1R), important for microglial survival, has suggested that ALAS is a microgliopathy. This study examines the correlation between microglial changes and axonopathy in ALAS. A total of 6 ALAS cases were studied. White matter lesions were classified into three evolving stages: 1) numerous axonal spheroids among well-myelinated fibers; 2) moderate loss of myelinated fibers with or without axonal spheroids; and 3) a leukodystrophy-like pattern of severe confluent axonal and myelin loss. Axonal spheroids and ramified microglia were semi-quantified and the lesions were assigned a score of 0-3. We found a strong correlation between the preponderance of axonal spheroids and ramified microglial loss. All areas with a predominance of axonal spheroids showed a near-complete absence of ramified microglia, which was also apparent in small cortical and white matter lesions. In contrast, some areas with no ramified microglia showed no axonal pathology. Our findings support the suggestion that ramified microglia loss precedes axonal spheroids formation. This observation will help to better understand the pathogenesis of ALAS and suggests a protective role of microglia.

Deep White Matter Lesions with Persistent Diffusion Restriction on MRI as a Diagnostic Clue: Neuroimaging of a Turkish Family with Hereditary Diffuse Leukoencephalopathy with Spheroids and Literature Review.

Background:Hereditary diffuse leukoencephalopathy with spheroids (HDLS), first described in 1984 is a rare disorder. Generally, it presents at adulthood with dementia, motor impairment, extrapyramidal abnormalities, and epilepsy. Definitive diagnosis is made by brain biopsy. Neuroimaging studies have revealed confluent white matter lesions predominantly in the frontal lobes, corpus callosum, and corticospinal tracts on conventional magnetic resonance imaging. Only a few reports showed diffusion restriction in the cerebral white matter; furthermore, rarer reports emphasized persistent foci of diffusion restriction as a diagnostic imaging marker.Objective:Herein, we have aimed to illustrate the first biopsy-proven Turkish HDLS pedigree consisting of 18 persons in 3 generations which contained 4 affected individuals.Materials and Methods:Four individuals in the pedigree of HDLS [two affected patients (patient III-1 and patient III-2) and two unaffected individuals (patient II-4 and patient III-5)] were investigated with conventional MRI and Diffusion-weighted imaging (DWI) using 1.5 Tesla (T) scanner. All four individuals were evaluated via neurological examinations and Mini-Mental State Examination. Brain biopsy study was performed on patient III-2. Finally, an extensive literature review involving pathology investigations and neuroimaging studies of HDLS patients was conducted.Results:DWIs of two investigated patients showed deep white matter lesions with persistent diffusion restriction. Computed tomography imaging showed punctate mineralization in the lesions. Biopsy specimens of patient III-2 demonstrated axonal spheroids which were typical for HDLS.Conclusions:Via the presentation of our pedigree and literature review, we suggest HDSL as a first-line differential diagnosis in patients with undiagnosed adult-onset familial leukoencephalopathy, in particular, those with MRI lesions of frontal white matter and centrum semiovale associated with foci of diffusion restriction and mineralization. Finally, we think that the persistence of the diffusion restriction in deep white matter lesions should be kept in mind as a crucial neuroimaging sign for HDLS.

Inhibiting store-operated calcium entry attenuates white matter secondary degeneration following SCI

Axonal degeneration plays a key role in the pathogenesis of numerous neurological disorders including spinal cord injury. After the irreversible destruction of the white matter elements during the primary (mechanical) injury, spared axons and their supporting glial cells begin to breakdown causing an expansion of the lesion site. Here we mechanistically link external sources of calcium entry through axoplasmic reticulum calcium store depletion that contributes to secondary axonal degeneration through a process called store-operated calcium entry. There is increasing evidence suggesting that store-operated calcium entry impairment is responsible for numerous disorders. Nevertheless, its role following spinal cord injury remains poorly understood. We hypothesize that store-operated calcium entry mediates secondary white matter degeneration after spinal cord injury. We used our previously published model of laser-induced spinal cord injury to focally transect mid cervical dorsal column axons from live 6–8-week-old heterozygous CNPaseGFP/+: Thy1YFP+ double transgenic murine spinal cord preparations (five treated, eight controls) and documented the dynamic changes in axons over time using two-photon excitation microscopy. We report that 1 hour delayed treatment with YM-58483, a potent inhibitor of store-operated calcium entry, significantly decreased intra-axonal calcium accumulation, axonal dieback both proximal and distal to the lesion site, reduced secondary axonal “bystander” damage acutely after injury, and promoted greater oligodendrocyte survival compared to controls. We also targeted store-operated calcium entry following a clinically relevant contusion spinal cord injury model in vivo. Adult, 6–8-week-old Advillin-Cre: Ai9 mice were subjected to a mild 30 kdyn contusion and imaged to observe secondary axonal degeneration in live animals. We found that delayed treatment with YM-58483 increased axonal survival and reduced axonal spheroid formation compared to controls (n = 5 mice per group). These findings suggest that blocking store-operated calcium entry acutely is neuroprotective and introduces a novel target to prevent pathological calcium entry following spinal cord injury using a clinically relevant model.

AAVrh10 Vector Corrects Disease Pathology in MPS IIIA Mice and Achieves Widespread Distribution of SGSH in Large Animal Brains

Patients with mucopolysaccharidosis type IIIA (MPS IIIA) lack the lysosomal enzyme sulfamidase (SGSH), which is responsible for the degradation of heparan sulfate (HS). Build-up of undegraded HS results in severe progressive neurodegeneration for which there is currently no treatment. The ability of the vector adeno-associated virus (AAV)rh.10-CAG-SGSH (LYS-SAF302) to correct disease pathology was evaluated in a mouse model for MPS IIIA. LYS-SAF302 was administered to 5-week-old MPS IIIA mice at three different doses (8.6E+08, 4.1E+10, and 9.0E+10 vector genomes [vg]/animal) injected into the caudate putamen/striatum and thalamus. LYS-SAF302 was able to dose-dependently correct or significantly reduce HS storage, secondary accumulation of GM2 and GM3 gangliosides, ubiquitin-reactive axonal spheroid lesions, lysosomal expansion, and neuroinflammation at 12 weeks and 25 weeks post-dosing. To study SGSH distribution in the brain of large animals, LYS-SAF302 was injected into the subcortical white matter of dogs (1.0E+12 or 2.0E+12 vg/animal) and cynomolgus monkeys (7.2E+11 vg/animal). Increases of SGSH enzyme activity of at least 20% above endogenous levels were detected in 78% (dogs 4 weeks after injection) and 97% (monkeys 6 weeks after injection) of the total brain volume. Taken together, these data validate intraparenchymal AAV administration as a promising method to achieve widespread enzyme distribution and correction of disease pathology in MPS IIIA.