Human Spinal Cord Tissue Research Articles

Integrated elemental analysis supports targeting copper perturbations as a therapeutic strategy in multiple sclerosis

Multiple sclerosis (MS) is a debilitating affliction of the central nervous system (CNS) that involves demyelination of neuronal axons and neurodegeneration resulting in disability that becomes more pronounced in progressive forms of the disease. The involvement of neurodegeneration in MS underscores the need for effective neuroprotective approaches necessitating identification of new therapeutic targets. Herein, we applied an integrated elemental analysis workflow to human MS-affected spinal cord tissue utilising multiple inductively coupled plasma-mass spectrometry methodologies. These analyses revealed shifts in atomic copper as a notable aspect of disease. Complementary gene expression and biochemical analyses demonstrated that changes in copper levels coincided with altered expression of copper handling genes and downstream functionality of cuproenzymes. Copper-related problems observed in the human MS spinal cord were largely reproduced in the experimental autoimmune encephalomyelitis (EAE) mouse model during the acute phase of disease characterised by axonal demyelination, lesion formation, and motor neuron loss. Treatment of EAE mice with the CNS-permeant copper modulating compound CuII(atsm) resulted in recovery of cuproenzyme function, improved myelination and lesion volume, and neuroprotection. These findings support targeting copper perturbations as a therapeutic strategy for MS with CuII(atsm) showing initial promise.

P.144 Comparative analysis of spinal cord-derived and induced pluripotent-derived neural stem & progenitor cells for SCI therapy

Background: Induced pluripotent stem cells (iPSCs) have revolutionized spinal cord injury (SCI) treatment by generating neural stem/progenitor cells (NSPCs). However, understanding how iPSC-derived NSPCs compare to authentic spinal cord NSPCs remains unclear. This study thoroughly characterizes bona fide spinal cord NSPCs and their isogenic iPSC-derived counterparts, iPSC-SC and iPSC-Br. Methods: Human spinal cord and skin tissue were obtained with ethics approval to establish primary NSPC cultures. iPSCs were derived from these primary cells and differentiated into iPSC-SC and iPSC-Br NSPCs. Assessments encompassed differentiation, proliferation capabilities, immunostaining, and RNA sequencing for differential gene expression. Results: Functional and transcriptional differences were identified between bona fide NSPCs and iPSC-SC/iPSC-Br. Bona fide and iPSC-SC NSPCs exhibited spinal cord regionalization, while iPSC-Br displayed forebrain regionalization. iPSC-derived NSPCs shared features reminiscent of early developmental stages, including embryonic patterning genes and increased proliferation rates. Notably, differentiation profiles were most similar between bona fide and iPSC-Br, with substantial distinctions observed between bona fide and iPSC-SC. Conclusions: This study unveils unique regional, developmental, and functional characteristics distinguishing spinal cord NSPCs from iPSC-derived counterparts. Addressing these disparities holds promise for enhancing iPSC-derived NSPC therapies in spinal cord injuries, contributing to a deeper understanding of their potential applications in regenerative medicine.

Constructing Linear-Oriented Pre-Vascularized Human Spinal Cord Tissues for Spinal Cord Injury Repair.

Repairing spinal cord injury (SCI) is a global medical challenge lacking effective clinical treatment. Developing human-engineered spinal cord tissues that can replenish lost cells and restore a regenerative microenvironment offers promising potential for SCI therapy. However, creating vascularized human spinal cord-like tissues (VSCT) that mimic the diverse cell types and longitudinal parallel structural features of spinal cord tissues remains a significant hurdle. In the present study, VSCTs are engineered using embryonic human spinal cord-derived neural and endothelial cells on linear-ordered collagen scaffolds (LOCS). Studies have shown that astrocytes and endothelial cells align along the scaffolds in VSCT, supporting axon extension from various human neurons myelinated by oligodendrocytes. After transplantation into SCI rats, VSCT survives at the injury sites and promotes endogenous neural regeneration and vascularization, ultimately reducing scarring and enhancing behavioral functional recovery. It suggests that pre-vascularization of engineered spinal cord tissues is beneficial for SCI treatment and highlights the important role of exogenous endothelial cells in tissue engineering.

Advanced Magnetic Resonance Imaging Biomarkers of the Injured Spinal Cord: A Comparative Study of Imaging and Histology in Human Traumatic Spinal Cord Injury.

A significant problem in the diagnosis and management of traumatic spinal cord injury (tSCI) is the heterogeneity of secondary injury and the prediction of neurological outcome. Imaging biomarkers specific to myelin loss and inflammation after tSCI would enable detailed assessment of the pathophysiological processes underpinning secondary damage to the cord. Such biomarkers could be used to biologically stratify injury severity and better inform prognosis for neurological recovery. While much work has been done to establish magnetic resonance imaging (MRI) biomarkers for SCI in animal models, the relationship between imaging findings and the underlying pathology has been difficult to discern in human tSCI because of the paucity of human spinal cord tissue. We utilized post-mortem spinal cords from individuals who had a tSCI to examine this relationship by performing ex vivo MRI scans before histological analysis. We investigated the correlation between the histological distribution of myelin loss and inflammatory cells in the injured spinal cord and a number of myelin and inflammation-sensitive MRI measures: myelin water fraction (MWF), inhomogeneous magnetization transfer ratio (ihMTR), and diffusion tensor and diffusion kurtosis imaging-derived fractional anisotropy (FA) and axial, radial, and mean diffusivity (AD, RD, MD). The histological features were analyzed by staining with Luxol Fast Blue (LFB) for myelin lipids and Class II major histocompatibility complex (Class II MHC) and CD68 for microglia and macrophages. Both MWF and ihMTR were strongly correlated with LFB staining for myelin, supporting the use of both as biomarkers for myelin loss after SCI. A decrease in ihMTR was also correlated with the presence of Class II MHC positive immune cells. FA and RD correlated with both Class II MHC and CD68 and may therefore be useful biomarkers for inflammation after tSCI. Our work demonstrates the utility of advanced MRI techniques sensitive to biological tissue damage after tSCI, which is an important step toward using these MRI techniques in the clinic to aid in decision-making.

Central nervous system sulfatide deficiency as a causal factor for bladder disorder in Alzheimer's disease.

Despite being a brain disorder, Alzheimer's disease (AD) is often accompanied by peripheral organ dysregulations (e.g., loss of bladder control in late-stage AD), which highly rely on spinal cord coordination. However, the causal factor(s) for peripheral organ dysregulation in AD remain elusive. The central nervous system (CNS) is enriched in lipids. We applied quantitative shotgun lipidomics to determine lipid profiles of human AD spinal cord tissues. Additionally, a CNS sulfatide (ST)-deficient mouse model was used to study the lipidome, transcriptome and peripheral organ phenotypes of ST loss. We observed marked myelin lipid reduction in the spinal cord of AD subjects versus cognitively normal individuals. Among which, levels of ST, a myelin-enriched lipid class, were strongly and negatively associated with the severity of AD. A CNS myelin-specific ST-deficient mouse model was used to further identify the causes and consequences of spinal cord lipidome changes. Interestingly, ST deficiency led to spinal cord lipidome and transcriptome profiles highly resembling those observed in AD, characterized by decline of multiple myelin-enriched lipid classes and enhanced inflammatory responses, respectively. These changes significantly disrupted spinal cord function and led to substantial enlargement of urinary bladder in ST-deficient mice. Our study identified CNS ST deficiency as a causal factor for AD-like lipid dysregulation, inflammation response and ultimately the development of bladder disorders. Targeting to maintain ST levels may serve as a promising strategy for the prevention and treatment of AD-related peripheral disorders.

Human spinal cord tissue is an underutilised resource in degenerative cervical myelopathy: findings from a systematic review of human autopsies

Study designSystematic review.BackgroundAlthough degenerative cervical myelopathy (DCM) is the most prevalent spinal cord condition worldwide, the pathophysiology remains poorly understood. Our objective was to evaluate existing histological findings of DCM on cadaveric human spinal cord tissue and explore their consistency with animal models.MethodsMEDLINE and Embase were systematically searched (CRD42021281462) for primary research reporting on histological findings of DCM in human cadaveric spinal cord tissue. Data was extracted using a piloted proforma. Risk of bias was assessed using Joanna Briggs Institute critical appraisal tools. Findings were compared to a systematic review of animal models (Ahkter et al. 2020 Front Neurosci 14).ResultsThe search yielded 4127 unique records. After abstract and full-text screening, 19 were included in the final analysis, reporting on 150 autopsies (71% male) with an average age at death of 67.3 years. All findings were based on haematoxylin and eosin (H&E) staining. The most commonly reported grey matter findings included neuronal loss and cavity formation. The most commonly reported white matter finding was demyelination. Axon loss, gliosis, necrosis and Schwann cell proliferation were also reported. Findings were consistent amongst cervical spondylotic myelopathy and ossification of the posterior longitudinal ligament. Cavitation was notably more prevalent in human autopsies compared to animal models.ConclusionFew human spinal cord tissue studies have been performed. Neuronal loss, demyelination and cavitation were common findings. Investigating the biological basis of DCM is a critical research priority. Human spinal cord specimen may be an underutilised but complimentary approach.

Diminished motor neuron activity driven by abnormal astrocytic EAAT1 glutamate transporter activity in spinal muscular atrophy is not fully restored after lentiviral SMN delivery.

Spinal muscular atrophy (SMA) is characterized by the loss of the lower spinal motor neurons due to survival motor neuron (SMN) deficiency. The motor neuron cell autonomous and non-cell autonomous disease mechanisms driving early glutamatergic dysfunction, a therapeutically targetable phenotype prior to motor neuron cell loss, remain unclear. Using microelectrode array analysis, we demonstrate that the secretome and cell surface proteins needed for proper synaptic modulation are likely disrupted in human SMA astrocytes and lead to diminished motor neuron activity. While healthy astrocyte conditioned media did not improve SMA motor neuron activity, SMA motor neurons robustly responded to healthy astrocyte neuromodulation in direct contact cultures. This suggests an important role of astrocyte synaptic-associated plasma membrane proteins and contact-mediated cellular interactions for proper motor neuron function in SMA. Specifically, we identified a significant reduction of the glutamate Na+ dependent excitatory amino acid transporter EAAT1 within human SMA astrocytes and SMA lumbar spinal cord tissue. The selective inhibition of EAAT1 in healthy co-cultures phenocopied the diminished neural activity observed in SMA astrocyte co-cultures. Caveolin-1, an SMN-interacting protein previously associated with local translation at the plasma membrane, was abnormally elevated in human SMA astrocytes. Although lentiviral SMN delivery to SMA astrocytes partially rescued EAAT1 expression, limited activity of healthy motor neurons was still observed in SMN-transduced SMA astrocyte co-cultures. Together, these data highlight the detrimental impact of astrocyte-mediated disease mechanisms on motor neuron function in SMA and that SMN delivery may be insufficient to fully restore astrocyte function at the synapse.

Generation of dorsoventral human spinal cord organoids via functionalizing composite scaffold for drug testing

The spinal cord possesses highly complex, finely organized cytoarchitecture guided by two dorsoventral morphogenic organizing centers. Thus, generation of human spinal cord tissue invitro is challenging. Here, we demonstrated a novel method for generation of human dorsoventral spinal cord organoids using composite scaffolds. Specifically, the spinal cord ventralizing signaling Shh agonist (SAG) was loaded into a porous chitosan microsphere (PCSM), then thermosensitive Matrigel was coated on the surface to form composite microspheres withfunctional sustained-release SAG, termed as PCSM-Matrigel@SAG. Using PCSM-Matrigel@SAG as the core to induce 3D engineering of human spinal cord organoids from human pluripotent stem cells (ehSC-organoids), we found ehSC-organoids could form dorsoventral spinal cord-like cytoarchitecture with major domain-specific progenitors and neurons. Besides, these ehSC-organoids also showed functional calcium activity. In summary, these ehSC-organoids are of great significance for modeling spinal cord development, drug screening as 3D models for motor neuron diseases, and spinal cord injury repair.

A C-Terminally Truncated TDP-43 Splice Isoform Exhibits Neuronal Specific Cytoplasmic Aggregation and Contributes to TDP-43 Pathology in ALS.

Neuronal cytoplasmic aggregation and ubiquitination of TDP-43 is the most common disease pathology linking Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 pathology is characterized by the presence of low molecular weight TDP-43 species generated through proteolytic cleavage and/or abnormal RNA processing events. In addition to N-terminally truncated TDP-43 species, it has become evident that C-terminally truncated variants generated through alternative splicing in exon 6 also contribute to the pathophysiology of ALS/FTLD. Three such variants are listed in UCSD genome browser each sharing the same C-terminal unique sequence of 18 amino acids which has been shown to contain a putative nuclear export sequence. Here we have identified an additional C-terminally truncated variant of TDP-43 in human spinal cord tissue. This variant, called TDP43C-spl, is generated through use of non-canonical splice sites in exon 6, skipping 1,020 bp and encoding a 272 aa protein lacking the C-terminus with the first 256 aa identical to full-length TDP-43 and the same 18 amino acid C-terminal unique sequence. Ectopic expression studies in cells revealed that TDP43C-spl was localized to the nucleus in astrocytic and microglial cell lines but formed cytoplasmic ubiquitinated aggregates in neuronal cell lines. An antibody raised to the unique 18 amino acid sequence showed elevated levels of C-terminally truncated variants in ALS spinal cord tissues, and co-labeled TDP-43 pathology in disease affected spinal motor neurons. The retention of this 18 amino acid sequence among several C-terminally truncated TDP-43 variants suggests important functional relevance. Our studies of TDP43C-spl suggest this may be related to the selective vulnerability of neurons to TDP-43 pathology and cell-subtype differences in nuclear export.

The role of aquaporin 4 (AQP4) in spinal cord injury.

Aquaporin-4 (AQP-4) is an aquaporin composed of six helical transmembrane domains and two highly conserved ASN-pro-ALA (NPA) motifs. It is strongly expressed in rodent and human spinal cord tissues and plays a key role in the pathological process after SCI. After SCI, edema, glial scarring, and inflammation can accelerate the progression of injury and lead to deterioration of function. Many studies have reported that AQP-4 plays an important role in SCI. In particular, it plays an important role in secondary pathological processes (spinal cord edema, glial scar formation, and inflammatory response) after SCI. Loss of AQP-4 has been associated with reduced spinal edema and improved prognosis after SCI in mice. In addition, downregulation of AQP-4 reduces glial scar formation and the inflammatory response after SCI. There is a consensus from numerous studies that AQP-4 may be a potential target for SCI therapy, which guides the ongoing investigation for molecular therapy of SCI. Here, we review the structure of AQP-4, its expression in normal and damaged spinal cord, and its role in SCI, as well as discuss the theoretical basis for the treatment of SCI.

K+/Cl- co-transporter-2 upmodulation: a multi-modal therapy to treat spinal cord injury.

K+/Cl- co-transporter-2 upmodulation: a multi-modal therapy to treat spinal cord injury.

Sex-dependent effects of amyloid precursor-like protein 2 in the SOD1-G37R transgenic mouse model of MND

Motor neurone disease (MND) is a neurodegenerative disorder characterised by progressive destruction of motor neurons, muscle paralysis and death. The amyloid precursor protein (APP) is highly expressed in the central nervous system and has been shown to modulate disease outcomes in MND. APP is part of a gene family that includes the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) genes. In the present study, we investigated the role of APLP2 in MND through the examination of human spinal cord tissue and by crossing APLP2 knockout mice with the superoxide dismutase 1 (SOD1-G37R) transgenic mouse model of MND. We found the expression of APLP2 is elevated in the spinal cord from human cases of MND and that this feature of the human disease is reproduced in SOD1-G37R mice at the End-stage of their MND-like phenotype progression. APLP2 deletion in SOD1-G37R mice significantly delayed disease progression and increased the survival of female SOD1-G37R mice. Molecular and biochemical analysis showed female SOD1-G37R:APLP2−/− mice displayed improved innervation of the neuromuscular junction, ameliorated atrophy of muscle fibres with increased APP protein expression levels in the gastrocnemius muscle. These results indicate a sex-dependent role for APLP2 in mutant SOD1-mediated MND and further support the APP family as a potential target for further investigation into the cause and regulation of MND.

Spinal Cord Injury Induces Permanent Reprogramming of Microglia into a Disease-Associated State Which Contributes to Functional Recovery.

Microglia are resident myeloid cells of the CNS. Recently, single-cell RNA sequencing (scRNAseq) has enabled description of a disease-associated microglia (DAM) with a role in neurodegeneration and demyelination. In this study, we use scRNAseq to investigate the temporal dynamics of immune cells harvested from the epicenter of traumatic spinal cord injury (SCI) induced in female mice. We find that as a consequence of SCI, baseline microglia undergo permanent transcriptional reprogramming into a previously uncharacterized subtype of microglia with striking similarities to previously reported DAM as well as a distinct microglial state found during development. Using a microglia depletion model we showed that DAM in SCI are derived from baseline microglia and strongly enhance recovery of hindlimb locomotor function following injury.SIGNIFICANCE STATEMENT Although disease-associated microglia (DAM) have been the subject of strong research interest during recent years (Keren-Shaul, 2017; Jordão, 2019), their cellular origin and their role in “normal” acute injury processes is not well understood. Our work directly addresses the origin and the role of DAM in traumatic injury response. Further, we use a microglia depletion model to prove that DAM in spinal cord injury (SCI) are indeed derived from homeostatic microglia, and that they strongly enhance recovery. Thus, in this work we significantly expand the knowledge of immune response to traumatic injury, demonstrate the applicability to human injury via our unique access to injured human spinal cord tissue, and provide the community with a comprehensive dataset for further exploration.

Native Separation and Metallation Analysis of SOD1 Protein from the Human Central Nervous System: a Methodological Workflow.

Studies of the metal content of metalloproteins in tissues from the human central nervous system (CNS) can be compromised by preparative techniques which alter levels of, or interactions between, metals and the protein of interest within a complex mixture. We developed a methodological workflow combining size exclusion chromatography, native isoelectric focusing, and either proton or synchrotron X-ray fluorescence within electrophoresis gels to analyze the endogenous metal content of copper-zinc superoxide dismutase (SOD1) purified from minimal amounts (<20 mg) of post-mortem human brain and spinal cord tissue. Abnormal metallation and aggregation of SOD1 are suspected to play a role in amyotrophic lateral sclerosis and Parkinson's disease, but data describing SOD1 metal occupancy in human tissues have not previously been reported. Validating our novel approach, we demonstrated step-by-step metal preservation, preserved SOD1 activity, and substantial enrichment of SOD1 protein versus confounding metalloproteins. We analyzed tissues from nine healthy individuals and five CNS regions (occipital cortex, substantia nigra, locus coeruleus, dorsal spinal cord, and ventral spinal cord). We found that Cu and Zn were bound to SOD1 in a ratio of 1.12 ± 0.28, a ratio very close to the expected value of 1. Our methodological workflow can be applied to the study of endogenous native SOD1 in a pathological context and adapted to a range of metalloproteins from human tissues and other sources.

Staufen1 in Human Neurodegeneration.

Mutations in the ATXN2 gene (CAG expansions ≥32 repeats) can be a rare cause of Parkinson's disease and amyotrophic lateral sclerosis (ALS). We recently reported that the stress granule (SG) protein Staufen1 (STAU1) was overabundant in neurodegenerative disorder spinocerebellar ataxia type 2 (SCA2) patient cells, animal models, and ALS-TDP-43 fibroblasts, and provided a link between SG formation and autophagy. We aimed to test if STAU1 overabundance has a role in the pathogenesis of other neurodegenerative diseases. With multiple neurodegenerative patient-derived cell models, animal models, and human postmortem ALS tissue, we evaluate STAU1 function using biochemical and immunohistological analyses. We demonstrate STAU1 overabundance and increased total and phosphorylated mammalian target of rapamycin (mTOR) in fibroblast cells from patients with ALS with mutations in TDP-43, patients with dementia with PSEN1 mutations, a patient with parkinsonism with MAPT mutation, Huntington's disease (HD) mutations, and SCA2 mutations. Increased STAU1 levels and mTOR activity were seen in human ALS spinal cord tissues as well as in animal models. Changes in STAU1 and mTOR protein levels were post-transcriptional. Exogenous expression of STAU1 in wildtype cells was sufficient to activate mTOR and downstream targets and form SGs. Targeting STAU1 by RNAi normalized mTOR, suggesting a potential role for therapy in diseases associated with STAU1 overabundance. STAU1 overabundance in neurodegeneration is a common phenomenon associated with hyperactive mTOR. Targeting STAU1 with ASOs or miRNA viral vectors may represent a novel, efficacious therapy for neurodegenerative diseases characterized by overabundant STAU1. ANN NEUROL 2021;89:1114-1128.

Identification and Preclinical Development of a 2,5,6-Trisubstituted Fluorinated Pyridine Derivative as a Radioligand for the Positron Emission Tomography Imaging of Cannabinoid Type 2 Receptors.

Despite the broad implications of the cannabinoid type 2 receptor (CB2) in neuroinflammatory processes, a suitable CB2-targeted probe is currently lacking in clinical routine. In this work, we synthesized 15 fluorinated pyridine derivatives and tested their binding affinities toward CB2 and CB1. With a sub-nanomolar affinity (Ki for CB2) of 0.8 nM and a remarkable selectivity factor of >12,000 over CB1, RoSMA-18-d6 exhibited outstanding in vitro performance characteristics and was radiofluorinated with an average radiochemical yield of 10.6 ± 3.8% (n = 16) and molar activities ranging from 52 to 65 GBq/μmol (radiochemical purity > 99%). [18F]RoSMA-18-d6 showed exceptional CB2 attributes as demonstrated by in vitro autoradiography, ex vivo biodistribution, and positron emission tomography (PET). Further, [18F]RoSMA-18-d6 was used to detect CB2 upregulation on postmortem human ALS spinal cord tissues. Overall, these results suggest that [18F]RoSMA-18-d6 is a promising CB2 PET radioligand for clinical translation.

A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells.

Improving the clinical translation of animal-based neural stem/progenitor cell (NSPC) therapies to humans requires an understanding of intrinsic human and animal cell characteristics. We report a novel in vitro method to assess spinal cord NSPCs from a small (rodent) and large (porcine) animal model in comparison to human NSPCs. To extract live adult human, porcine, and rodent spinal cord tissue, we illustrate a strategy using an anterior or posterior approach that was simulated in a porcine model. The initial expansion of primary NSPCs is carried out using the neurosphere assay followed by a pharmacological treatment phase during which NSPCs derived from humans, porcines, and rodents are assessed in parallel using the same defined parameters. Using this model, NSPCs from all species demonstrated multi-lineage differentiation and self-renewal. Importantly, these methods provide conditions to enable the direct comparison of species-dependent cell behavior in response to specific exogenous signals.

Spatiotemporal development of spinal neuronal and glial populations in the Ts65Dn mouse model of Down syndrome

BackgroundDown syndrome (DS), caused by the triplication of chromosome 21, results in a constellation of clinical features including changes in intellectual and motor function. Although altered neural development and function have been well described in people with DS, few studies have investigated the etiology underlying the observed motor phenotypes. Here, we examine the development, patterning, and organization of the spinal cord throughout life in the Ts65Dn mouse, a model that recapitulates many of the motor changes observed in people with DS.MethodsSpinal cords from embryonic to adult animals were processed for gene and protein expression (immunofluorescence) to track the spatiotemporal development of excitatory and inhibitory neurons and oligodendroglia. Postnatal analyses were focused on the lumbar region due to the reflex and gait abnormalities found in Ts65Dn mice and locomotive alterations seen in people with DS.ResultsBetween embryonic days E10.5 and E14.5, we found a larger motor neuron progenitor domain in Ts65Dn animals containing more OLIG2-expressing progenitor cells. These disturbed progenitors are delayed in motor neuron production but eventually generate a large number of ISL1+ migrating motor neurons. We found that higher numbers of PAX6+ and NKX2.2+ interneurons (INs) are also produced during this time frame. In the adult lumbar spinal cord, we found an increased level of Hb9 and a decreased level of Irx3 gene expression in trisomic animals. This was accompanied by an increase in Calretinin+ INs, but no changes in other neuronal populations. In aged Ts65Dn animals, both Calbindin+ and ChAT+ neurons were decreased compared to euploid controls. Additionally, in the dorsal corticospinal white matter tract, there were significantly fewer CC1+ mature OLs in 30- and 60-day old trisomic animals and this normalized to euploid levels at 10–11 months. In contrast, the mature OL population was increased in the lateral funiculus, an ascending white matter tract carrying sensory information. In 30-day old animals, we also found a decrease in the number of nodes of Ranvier in both tracts. This decrease normalized both in 60-day old and aged animals.ConclusionsWe show marked changes in both spinal white matter and neuronal composition that change regionally over the life span. In the embryonic Ts65Dn spinal cord, we observe alterations in motor neuron production and migration. In the adult spinal cord, we observe changes in oligodendrocyte maturation and motor neuron loss, the latter of which has also been observed in human spinal cord tissue samples. This work uncovers multiple cellular perturbations during Ts65Dn development and aging, many of which may underlie the motor deficits found in DS.

Structure-Activity Relationship Studies of Pyridine-Based Ligands and Identification of a Fluorinated Derivative for Positron Emission Tomography Imaging of Cannabinoid Type 2 Receptors.

The cannabinoid type 2 (CB2) receptor has emerged as a valuable target for therapy and imaging of immune-mediated pathologies. With the aim to find a suitable radiofluorinated analogue of the previously reported CB2 positron emission tomography (PET) radioligand [11C]RSR-056, 38 fluorinated derivatives were synthesized and tested by in vitro binding assays. With a Ki (hCB2) of 6 nM and a selectivity factor of nearly 700 over cannabinoid type 1 receptors, target compound 3 exhibited optimal in vitro properties and was selected for evaluation as a PET radioligand. [18F]3 was obtained in an average radiochemical yield of 11 ± 4% and molar activities between 33 and 114 GBq/μmol. Specific binding of [18F]3 to CB2 was demonstrated by in vitro autoradiography and in vivo PET experiments using the CB2 ligand GW-405 833. Metabolite analysis revealed only intact [18F]3 in the rat brain. [18F]3 detected CB2 upregulation in human amyotrophic lateral sclerosis spinal cord tissue and may thus become a candidate for diagnostic use in humans.

Proteomics in cerebrospinal fluid and spinal cord suggests UCHL1, MAP2 and GPNMB as biomarkers and underpins importance of transcriptional pathways in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease and the proteins and pathways involved in the pathophysiology are not fully understood. Even less is known about the preclinical disease phase. To uncover new ALS-related proteins and pathways, we performed a comparative proteomic analysis in cerebrospinal fluid (CSF) of asymptomatic (n = 14) and symptomatic (n = 14) ALS mutation carriers and sporadic ALS patients (n = 12) as well as post-mortem human spinal cord tissue (controls: n = 7, ALS, n = 8). Using a CSF-optimized proteomic workflow, we identified novel (e.g., UCHL1, MAP2, CAPG, GPNMB, HIST1H4A, HIST1H2B) and well-described (e.g., NEFL, NEFH, NEFM, CHIT1, CHI3L1) protein level changes in CSF of sporadic and genetic ALS patients with enrichment of proteins related to transcription, cell cycle and lipoprotein remodeling (total protein IDs: 2303). No significant alteration was observed in asymptomatic ALS mutation carriers representing the prodromal disease phase. We confirmed UCHL1, MAP2, CAPG and GPNMB as novel biomarker candidates for ALS in an independent validation cohort of patients (n = 117) using multiple reaction monitoring. In spinal cord tissue, 292 out of 6810 identified proteins were significantly changed in ALS with enrichment of proteins involved in mRNA splicing and of the neurofilament compartment. In conclusion, our proteomic data in asymptomatic ALS mutation carriers support the hypothesis of a sudden disease onset instead of a long preclinical phase. Both CSF and tissue proteomic data indicate transcriptional pathways to be amongst the most affected. UCHL1, MAP2 and GPNMB are promising ALS biomarker candidates which might provide additional value to the established neurofilaments in patient follow-up and clinical trials.