Neurofilament Phosphorylation Research Articles

The dynamic of changes of pNFH levels in the CSF compared with the motor scales’ scores during three years of nusinersen treatment in children with spinal muscular atrophy types 2 and 3

Abstract: Neurofilaments are crucial in neuronal cytoskeleton formation, influencing axonal growth and impulse modulation. This study focuses on understanding the dynamics of the phos-phorylated neurofilament heavy subunit (pNFH) in pediatric spinal muscular atrophy (SMA) pa-tients undergoing Nusinersen treatment. The presence of five neurofilament types, particularly pNFH, is explored as a potential biomarker. SMA, an autosomal recessive disease impacting motor neurons, is characterized by disease severity linked to the number of SMN2 gene copies. Approved drugs, including Nusinersen, have demonstrated efficacy in enhancing motor activity. Methods: A retrospective analysis was conducted on 18 pediatric SMA patients treated with Nusinersen from October 2018 to July 2023. Cerebrospinal fluid (CSF) samples were utilized to assess pNFH levels. Motor scales were employed to evaluate performance, focusing on patients with varying SMN2 gene copies. Results: Following the initiation of Nusinersen treatment, a substantial decrease in pNFH levels was observed in CSF samples. Motor scales indicated improved performance, partic-ularly in patients with more SMN2 copies. However, the correlation between pNFH levels and motor improvement was not strongly evident, suggesting a limited role as a prognostic indicator within this timeframe. Conclusion: Nusinersen effectively reduced pNFH levels in pediatric SMA patients, showcasing promising outcomes in motor function. However, the predictive value of pNFH remains inconclusive, emphasizing the need for further research. Study limitations, including the rarity of SMA, the absence of a control group, and the disease's dynamic nature over time, should be considered when interpreting these findings.

A novel HSPB1S139F mouse model of Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth Disease (CMT) is a commonly inherited peripheral polyneuropathy. Clinical manifestations for this disease include symmetrical distal polyneuropathy, altered deep tendon reflexes, distal sensory loss, foot deformities, and gait abnormalities. Genetic mutations in heat shock proteins have been linked to CMT2. Specifically, mutations in the heat shock protein B1 (HSPB1) gene encoding for heat shock protein 27 (Hsp27) have been linked to CMT2F and distal hereditary motor and sensory neuropathy type 2B (dHMSN2B) subtype. The goal of the study was to examine the role of an endogenous mutation in HSPB1 in vivo and to define the effects of this mutation on motor function and pathology in a novel animal model. As sphingolipids have been implicated in hereditary and sensory neuropathies, we examined sphingolipid metabolism in central and peripheral nervous tissues in 3-month-old HspS139F mice. Though sphingolipid levels were not altered in sciatic nerves from HspS139F mice, ceramides and deoxyceramides, as well as sphingomyelins (SMs) were elevated in brain tissues from HspS139F mice. Histology was utilized to further characterize HspS139F mice. HspS139F mice exhibited no alterations to the expression and phosphorylation of neurofilaments, or in the expression of acetylated α-tubulin in the brain or sciatic nerve. Interestingly, HspS139F mice demonstrated cerebellar demyelination. Locomotor function, grip strength and gait were examined to define the role of HspS139F in the clinical phenotypes associated with CMT2F. Gait analysis revealed no differences between HspWT and HspS139F mice. However, both coordination and grip strength were decreased in 3-month-old HspS139F mice. Together these data suggest that the endogenous S139F mutation in HSPB1 may serve as a mouse model for hereditary and sensory neuropathies such as CMT2F.

Involvement of casein kinase 1 epsilon/delta (Csnk1e/d) in the pathogenesis of familial Parkinson's disease caused by CHCHD2.

Parkinson's disease (PD) is a common neurodegenerative disorder that results from the loss of dopaminergic neurons. Mutations in coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) gene cause a familial form of PD with α-Synuclein aggregation, and we here identified the pathogenesis of the T61I mutation, the most common disease-causing mutation of CHCHD2. In Neuro2a cells, CHCHD2 is in mitochondria, whereas the T61I mutant (CHCHD2T61I ) is mislocalized in the cytosol. CHCHD2T61l then recruits casein kinase 1 epsilon/delta (Csnk1e/d), which phosphorylates neurofilament and α-Synuclein, forming cytosolic aggresomes. In vivo, both Chchd2T61I knock-in and transgenic mice display neurodegenerative phenotypes and aggresomes containing Chchd2T61I , Csnk1e/d, phospho-α-Synuclein, and phospho-neurofilament in their dopaminergic neurons. Similar aggresomes were observed in a postmortem PD patient brain and dopaminergic neurons generated from patient-derived iPS cells. Importantly, a Csnk1e/d inhibitor substantially suppressed the phosphorylation of neurofilament and α-Synuclein. The Csnk1e/d inhibitor also suppressed the cellular damage in CHCHD2T61I -expressing Neuro2a cells and dopaminergic neurons generated from patient-derived iPS cells and improved the neurodegenerative phenotypes of Chchd2T61I mutant mice. These results indicate that Csnk1e/d is involved in the pathogenesis of PD caused by the CHCHD2T61I mutation.

Review of Neurofilaments as Biomarkers in Sepsis-Associated Encephalopathy.

Sepsis is a common and fatal disease, especially in critically ill patients. Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction with acute altered consciousness, permanent cognitive impairment, and even coma, accompanied by sepsis, without direct central nervous system infection. When managing SAE, early identification and quantification of axonal damage facilitate faster and more accurate diagnosis and prognosis. Although no specific markers for SAE have been identified, several biomarkers have been proposed. Neurofilament light chain (NFL) is a highly expressed cytoskeletal component of neurofilament (NF) proteins that can be found in blood and cerebrospinal fluid (CSF) after exposure to axonal injury. NFs can be used as diagnostic and prognostic biomarkers for sepsis-related brain injury. Phosphorylation of NFs contributes to the maturation and stabilization of cytoskeletal structures, especially axons, and facilitates axonal transport, including mitochondrial transport and energy transport. The stability of NF proteins can be assessed by monitoring the expression of NF genes. Furthermore, phosphorylation levels of NFs can be monitored to determine mitochondrial axonal transport associated with cellular energy metabolism at distal axons to assess progression during SAE treatment. This paper provides new insights into the biological characteristics, detection techniques, and scientific achievements of NFs, and discusses the underlying mechanisms and future research directions of NFs in SAE.

How does neurofilament phosphorylation regulate axonal outgrowth and stabilization?

Neurofilaments (NFs) are tissue-specific intermediate filaments that maintain neuronal structural stability. Mammalian NFs are composed of Heavy (NF-H), Medium (NF-M), and Light (NF-L) subunits, named according to their molecular mass, along with α-internexin or peripherin (in central or peripheral nervous systems, respectively). NF subunits assemble linearly, with C-terminal regions extending from the filament “backbone.” Phosphorylation of C-terminal “side arms” of NF-H promotes NF-NF interactions, providing stability to axons.

Diffuse Traumatic Injury in the Mouse Disrupts Axon-Myelin Integrity in the Cerebellum.

Cerebellar dysfunction after traumatic brain injury (TBI) is commonly suspected based on clinical symptoms, although cerebellar pathology has rarely been investigated. To address the hypothesis that the cerebellar axon-myelin unit is altered by diffuse TBI, we used the central fluid percussion injury (cFPI) model in adult mice to create widespread axonal injury by delivering the impact to the forebrain. We specifically focused on changes in myelin components (myelin basic protein [MBP], 2',3'-cyclic nucleotide 3'-phosphodiesterase [CNPase], nodal/paranodal domains [neurofascin (Nfasc), ankyrin-G], and phosphorylated neurofilaments [SMI-31, SMI-312]) in the cerebellum, remote from the impact, at two, seven, and 30 days post-injury (dpi). When compared with sham-injured controls, cerebellar MBP and CNPase protein levels were decreased at 2 dpi that remained reduced up to 30 dpi. Diffuse TBI induced different effects on neuronal (Nfasc 186, Nfasc 140) and glial (Nfasc 155) neurofascin isoforms that play a key role in the assembly of the nodes of Ranvier. Expression of Nfasc 140 in the cerebellum increased at 7 dpi, in contrast to Nfasc 155 levels, which were decreased. Although neurofascin binding partner ankyrin-G protein levels decreased acutely after cFPI, its expression levels increased at 7 dpi and remained unchanged up to 30 dpi. The TBI-induced reduction in neurofilament phosphorylation (SMI-31) observed in the cerebellum was closely associated with decreased levels of the myelin proteins MBP and CNPase. This is the first evidence of temporal and spatial structural changes in the axon-myelin unit in the cerebellum, remote from the location of the impact site, in a diffuse TBI model in mice.

Associations of fully automated Elecsys CSF and novel plasma biomarkers with Alzheimer's disease neuropathology

AbstractBackgroundThe development of fully automated analysis platforms for cerebrospinal fluid (CSF) biomarkers of Alzheimer’s disease (AD) and recent advances in blood‐derived biomarkers signify major steps towards a widespread clinical use of fluid‐based biomarkers. We aimed to study AD CSF biomarkers analyzed by fully automated Elecsys immunoassays in comparison to neuropathologic gold standards, and compare their accuracy to plasma phosphorylated tau (p‐tau181) and neurofilament light (NfL) measured using novel Simoa assays.MethodWe compared ante‐mortem Elecsys‐derived CSF biomarkers to standardised post‐mortem assessments of AD and non‐AD neuropathologic changes in 45 participants from the Alzheimer’s Disease Neuroimaging Initiative autopsy cohort. In a subset of 26 participants, we also analyzed ante‐mortem levels of plasma p‐tau181 and NfL. Reference biomarker values were obtained from 146 amyloid‐PET‐negative healthy controls (HC)([18F]Florbetapir‐PET <12 Centiloids).ResultAll CSF biomarkers clearly distinguished pathology‐confirmed AD dementia patients (N=27) from HC (AUCs=0.86‐1.00). CSF total‐tau (t‐tau), p‐tau181, and their ratios with Aβ1‐42, also accurately distinguished pathology‐confirmed AD from non‐AD dementia (N=8; AUCs=0.94‐0.97)(Fig. 1). In pathology‐specific analyses, intermediate‐to‐high Thal amyloid phases were best detected by CSF Aβ1‐42 (AUC[95% CI]=0.91[0.81‐1]), while intermediate‐to‐high CERAD neuritic plaques and Braak tau stages were best detected by CSF p‐tau181 (AUC=0.89[0.79‐0.99] and 0.88[0.77‐0.99], respectively)(Fig. 2). Optimal Elecsys CSF biomarker cut‐offs were derived at 1097/229/19 pg/ml for Aβ1‐42, t‐tau, and p‐tau181. In the plasma subsample, both plasma p‐tau181 (AUC=0.91[0.86‐0.96]) and NfL (AUC=0.93[0.87‐0.99]) accurately distinguished pathology‐confirmed AD (N=14) from HC. However, only p‐tau181 distinguished AD from non‐AD dementia cases (N=4; AUC=0.96[0.88‐1.00])(Fig. 1), and showed a similar, though weaker, pathologic specificity for neuritic plaques (AUC=0.75[0.52‐0.98]) and Braak stage (AUC=0.71[0.44‐0.98]) as CSF p‐tau181. No CSF or plasma biomarker was sensitive to the presence of Lewy body or TDP‐43 pathology, but Elecsys CSF Aβ1‐42 levels detected the presence of cerebral amyloid angiopathy (AUC=0.84[0.73‐0.96]).ConclusionWe demonstrate for the first time that the fully automated Elecsys CSF biomarkers detect AD neuropathologic changes with very high discriminative accuracy in vivo, and we report corresponding pathology‐based cut‐offs for these standardized biomarker measurements. Preliminary findings in a smaller subsample also support the use of plasma p‐tau181 as an easily accessible and scalable biomarker of AD pathology.

A possible mechanism for neurofilament slowing down in myelinated axon: Phosphorylation-induced variation of NF kinetics.

Neurofilaments(NFs) are the most abundant intermediate filaments that make up the inner volume of axon, with possible phosphorylation on their side arms, and their slow axonal transport by molecular motors along microtubule tracks in a “stop-and-go” manner with rapid, intermittent and bidirectional motion. The kinetics of NFs and morphology of axon are dramatically different between myelinate internode and unmyelinated node of Ranvier. The NFs in the node transport as 7.6 times faster as in the internode, and the distribution of NFs population in the internode is 7.6 folds as much as in the node of Ranvier. We hypothesize that the phosphorylation of NFs could reduce the on-track rate and slow down their transport velocity in the internode. By modifying the ‘6-state’ model with (a) an extra phosphorylation kinetics to each six state and (b) construction a new ‘8-state’ model in which NFs at off-track can be phosphorylated and have smaller on-track rate, our model and simulation demonstrate that the phosphorylation-induced decrease of on-track rate could slow down the NFs average velocity and increase the axonal caliber. The degree of phosphorylation may indicate the extent of velocity reduction. The Continuity equation used in our paper predicts that the ratio of NFs population is inverse proportional to the ratios of average velocity of NFs between node of Ranvier and internode. We speculate that the myelination of axon could increase the level of phosphorylation of NF side arms, and decrease the possibility of NFs to get on-track of microtubules, therefore slow down their transport velocity. In summary, our work provides a potential mechanism for understanding the phosphorylation kinetics of NFs in regulating their transport and morphology of axon in myelinated axons, and the different kinetics of NFs between node and internode.

Cerebellar cortical degeneration with increased phosphorylated neurofilament heavy chain in cerebrospinal fluid in a Holstein calf

Cerebellar cortical degeneration with increased phosphorylated neurofilament heavy chain in cerebrospinal fluid in a Holstein calf

Neurotrophic effects of GM1 ganglioside, NGF, and FGF2 on canine dorsal root ganglia neurons in vitro

Dogs share many chronic morbidities with humans and thus represent a powerful model for translational research. In comparison to rodents, the canine ganglioside metabolism more closely resembles the human one. Gangliosides are components of the cell plasma membrane playing a role in neuronal development, intercellular communication and cellular differentiation. The present in vitro study aimed to characterize structural and functional changes induced by GM1 ganglioside (GM1) in canine dorsal root ganglia (DRG) neurons and interactions of GM1 with nerve growth factor (NGF) and fibroblast growth factor (FGF2) using immunofluorescence for several cellular proteins including neurofilaments, synaptophysin, and cleaved caspase 3, transmission electron microscopy, and electrophysiology. GM1 supplementation resulted in increased neurite outgrowth and neuronal survival. This was also observed in DRG neurons challenged with hypoxia mimicking neurodegenerative conditions due to disruptions of energy homeostasis. Immunofluorescence indicated an impact of GM1 on neurofilament phosphorylation, axonal transport, and synaptogenesis. An increased number of multivesicular bodies in GM1 treated neurons suggested metabolic changes. Electrophysiological changes induced by GM1 indicated an increased neuronal excitability. Summarized, GM1 has neurotrophic and neuroprotective effects on canine DRG neurons and induces functional changes. However, further studies are needed to clarify the therapeutic value of gangliosides in neurodegenerative diseases.

DPP-4 inhibitor improves learning and memory deficits and AD-like neurodegeneration by modulating the GLP-1 signaling

Glucagon-like peptide-1 (GLP-1) signaling in the brain plays an important role in the regulation of glucose metabolism, which is impaired in Alzheimer's disease (AD). Here, we detected the GLP-1 and GLP-1 receptor (GLP-1R) in AD human brain and APP/PS1/Tau transgenic (3xTg) mice brain, finding that they were both decreased in AD human and mice brain. Enhanced GLP-1 exerts its protective effects on AD, however, this is rapidly degraded into inactivated metabolites by dipeptidyl peptidase-4 (DPP-4), resulting in its extremely short half-time. DPP-4 inhibitors, thus, was applied to improve the level of GLP-1 and GLP-1R expression in the hippocampus and cortex of AD mice brains. It is also protected learning and memory and synaptic proteins, increased the O-Glycosylation and decreased abnormal phosphorylation of tau and neurofilaments (NFs), degraded intercellular β-amyloid (Aβ) accumulation and alleviated neurodegeneration related to GLP-1 signaling pathway.

Alteration of O-GlcNAcylation affects assembly and axonal transport of neurofilament via phosphorylation

Neurofilaments (NFs), the most abundant cytoskeletal components in the mature neuron, are hyperphosphorylated and accumulated in the neuronal cell body of AD brain, and the abnormalities of NFs appear to contribute to neurodegeneration. Although previous studies have showed that O-GlcNAcylation and phosphorylation of NFs regulate each other reciprocally, the NFs O-GlcNAcylation and its effects on assembly and axonal transport are poorly explored. Here, we focus on the role of dysregulation of O-GlcNAcylation on structure and function of neurofilaments by corresponding phosphorylation.In the study, we found that decreased O-GlcNAcylation by intracerebroventricular administration of Alloxan, 6-diazo-5-oxonorleucine (Don) and okadaic acid (OA) in the rats resulted in increased phosphorylation with assembly of lower and shorter NFs. In contrast, in the sample of NAG-thiazoline (NAG-Ae) causing increased O-GlcNAcylation, NFs showed elongated filaments fibers and higher proportion of assembly. Furthermore, alloxan treatment induced abnormal accumulation of NFs bodies and delayed time of Fluorescence Recovery After Photobleaching (FRAP) in SK-N-SH cells, but the NAG-Ae treatment speeded up the axonal transport.Our experiments suggest that increased O-GlcNAcylation plays a key role in protecting the structure and function of NFs including filament assembly and axonal transport via decreased phosphorylation. These results expanded the function of O-GlcNAcylation in AD pathogenesis.

The sympathetic nervous system regulates skeletal muscle motor innervation and acetylcholine receptor stability.

Symptoms of autonomic failure are frequently the presentation of advanced age and neurodegenerative diseases that impair adaptation to common physiologic stressors. The aim of this work was to examine the interaction between the sympathetic and motor nervous system, the involvement of the sympathetic nervous system (SNS) in neuromuscular junction (NMJ) presynaptic motor function, the stability of postsynaptic molecular organization, and the skeletal muscle composition and function. Since muscle weakness is a symptom of diseases characterized by autonomic dysfunction, we studied the impact of regional sympathetic ablation on muscle motor innervation by using transcriptome analysis, retrograde tracing of the sympathetic outflow to the skeletal muscle, confocal and electron microscopy, NMJ transmission by electrophysiological methods, protein analysis, and state of the art microsurgical techniques, in C57BL6, MuRF1KO and Thy-1 mice. We found that the SNS regulates motor nerve synaptic vesicle release, skeletal muscle transcriptome, muscle force generated by motor nerve activity, axonal neurofilament phosphorylation, myelin thickness, and myofibre subtype composition and CSA. The SNS also modulates the levels of postsynaptic membrane acetylcholine receptor by regulating the Gαi2 -Hdac4-Myogenin-MuRF1pathway, which is prevented by the overexpression of the guanine nucleotide-binding protein Gαi2 (Q205L), a constitutively active mutant G protein subunit. The SNS regulates NMJ transmission, maintains optimal Gαi2 expression, and prevents any increase in Hdac4, myogenin, MuRF1, and miR-206. SNS ablation leads to upregulation of MuRF1, muscle atrophy, and downregulation of postsynaptic AChR. Our findings are relevant to clinical conditions characterized by progressive decline of sympathetic innervation, such as neurodegenerative diseases and aging.

Neuroplastic change of cytoskeleton in inferior colliculus after auditory deafferentation

Neural plasticity is a characteristic of the brain that helps it adapt to changes in sensory input. We hypothesize that auditory deafferentation may induce plastic changes in the cytoskeleton of the neurons in the inferior colliculus (IC). In this study, we evaluated the dynamic status of neurofilament (NF) phosphorylation in the IC after hearing loss. We induced auditory deafferentation via unilateral or bilateral cochlear ablation in rats, aged 4 weeks. To evaluate cytoskeletal changes in neurons, we evaluated mRNA fold changes in NF heavy chain expression, non-phosphorylated NF protein fold changes using SMI-32 antibody, and the ratio of SMI-32 immunoreactive (SMI-32-ir) neurons to the total neuronal population in the IC at 4 and 12 weeks after deafness. In the bilateral deafness (BD) group, the ratios of SMI-32-ir neurons significantly increased at 4 weeks after ablation in the right and left IC (6.1 ± 4.4%, 5.0 ± 3.4%, respectively), compared with age-matched controls (P < 0.01, P < 0.01). At 12 weeks after ablation, the ratio of SMI-32 positive neurons was higher (right, 3.4 ± 2.0%; left, 3.2 ± 2.3%) than that in the age-matched control group, albeit not significant in the right and left side (P = 0.38, P = 0.24, respectively). Consistent with the results of the ratio of SMI-32-ir neurons, SMI-32-ir protein expression was increased at 4 weeks after BD, and the changes at 12 weeks after bilateral ablation were not significant in the right or left IC. The age-matched control fold changes of NF mRNA expression after bilateral deafness were not significant at 4 and 12 weeks after deafness in right and left IC. Unilateral deafness did not induce significant change of NF mRNA expression, SMI-32-ir protein expression, and the ratio of SMI-32-ir neurons in the IC at 4 and 12 weeks after hearing loss. Bilateral auditory deafferentation induces structural changes in the neuronal cytoskeleton within the IC, which is prominent at 4 weeks after BD. The structural remodeling of neurons stabilized at 12 weeks after BD. Unlike BD, unilateral auditory deafferentation did not affect the dynamic status of NFs in the IC.

Cerebrospinal Fluid from Patients with Sporadic Amyotrophic Lateral Sclerosis Induces Degeneration of Motor Neurons Derived from Human Embryonic Stem Cells.

Disease modeling has become challenging in the context of amyotrophic lateral sclerosis (ALS), as obtaining viable spinal motor neurons from postmortem patient tissue is an unlikely possibility. Limitations in the animal models due to their phylogenetic distance from human species hamper the success of translating possible findings into therapeutic options. Accordingly, there is a need for developing humanized models as a lead towards identifying successful therapeutic possibilities. In this study, human embryonic stem cells-BJNHem20-were differentiated into motor neurons expressing HB9, Islet1, and choline acetyl transferase using retinoic acid and purmorphamine. These motor neurons discharged spontaneous action potentials with two different frequencies (< 5 and > 5Hz), and majority of them were principal neurons firing with < 5Hz. Exposure to cerebrospinal fluid from ALS patients for 48h induced several degenerative changes in the motor neurons as follows: cytoplasmic changes such as beading of neurites and vacuolation; morphological alterations, viz., dilation and vacuolation of mitochondria, curled and closed Golgi architecture, dilated endoplasmic reticulum, and chromatin condensation in the nucleus; lowered activity of different mitochondrial complex enzymes; reduced expression of brain-derived neurotrophic factor; up-regulated neurofilament phosphorylation and hyperexcitability represented by increased number of spikes. All these changes along with the enhanced expression of pro-apoptotic proteins-Bax and caspase 9-culminated in the death of motor neurons.

Internode length is reduced during myelination and remyelination by neurofilament medium phosphorylation in motor axons

The distance between nodes of Ranvier, referred to as internode length, positively correlates with axon diameter, and is optimized during development to ensure maximal neuronal conduction velocity. Following myelin loss, internode length is reestablished through remyelination. However, remyelination results in short internode lengths and reduced conduction rates. We analyzed the potential role of neurofilament phosphorylation in regulating internode length during remyelination and myelination. Following ethidium bromide induced demyelination, levels of neurofilament medium (NF-M) and heavy (NF-H) phosphorylation were unaffected. Preventing NF-M lysine-serine-proline (KSP) repeat phosphorylation increased internode length by 30% after remyelination. To further analyze the role of NF-M phosphorylation in regulating internode length, gene replacement was used to produce mice in which all KSP serine residues were replaced with glutamate to mimic constitutive phosphorylation. Mimicking constitutive KSP phosphorylation reduced internode length by 16% during myelination and motor nerve conduction velocity by ~27% without altering sensory nerve structure or function. Our results suggest that NF-M KSP phosphorylation is part of a cooperative mechanism between axons and Schwann cells that together determine internode length, and suggest motor and sensory axons utilize different mechanisms to establish internode length.

Evaluation Of Electrophysiological Changes In Primary Spinal Cord Neuronal Cultures Exposed To ALS-CSF Using Microelectrode Array.

Evaluation Of Electrophysiological Changes In Primary Spinal Cord Neuronal Cultures Exposed To ALS-CSF Using Microelectrode Array.

Multiple Sclerosis: Neurofilament Pathology in Spinal Motor Neurons

Objective: While traditionally a disorder of myelin, in multiple sclerosis (MS) neuronal and axonal damage has in recent years become an important topic of clinical relevance. To address this, alterations in neurofilament phosphorylation, known markers of neuronal health, were investigated in anterior horn cells in MS spinal cord tissue for signs of motor neuron damage. Methods: Spinal cord tissue was examined from 13 MS and 6 control patients. Fresh frozen sections were labelled with antibodies against phosphorylated and non-phosphorylated epitopes of neurofilament H (NF-H) and analyzed by light microscopy. Results: In MS, increased expression of phosphorylated NF-H in spinal motor neuron perikarya (abnormal for neurons) occurred in 61.5% of cases, mostly in chronic active lesions, with the strongest immunoreactivity at the lumbar level. Inflammatory activity was common in chronic active but rare in chronic silent lesions. In one case with an acute lesion, we saw swollen axons positive for non-phosphorylated NF-H, a pathologic marker in axons, but no signs of perikaryal damage. Expression of non-phosphorylated NF-H in spinal motor neuron perikarya was similar in both MS and controls. Conclusion: In line with previous studies, our findings implicate anterior horn cell damage as a common feature in MS. We propose that underlying mechanisms may involve reduced synaptic input and/or retrograde degeneration, subjects which remain to be investigated.

383 - Sulfurous Heterocycles Based on the Transsulfuration Metabolite Lanthionine Ketimine Are Potent, Selective Antagonists of Cdk5/p25 Signaling with Broad Potential to Treat Neurodegenerative Diseases

Lanthionine ketimine (LK) is an endogenous amino acid metabolite thought to form when promiscuous reactions of the transsulfuration pathway create thioether amino acid intermediates that undergo further transamination reactions catalyzed mostly by glutamine transaminase K (kynurenine aminotransferase). Cell penetrating ester derivatives of LKE have potent antioxidant, anti-neuroinflammatory and pro-neurotrophic abilities and reduce correlates of disease in diverse animal models of neurodegenerative pathologies. The precise mechanism of action for LK derivatives has remained elusive though LK-ethyl ester (LKE, also known as XN001) has proven to be a potent enhancer of cell autophagy in cell culture and in vivo. In the course of translational development of XN001 for neurodisease therapy, data was uncovered that this compound potently and selectively inhibits the cyclin dependent kinase-5(Cdk5)/p25 complex with no inhibition of Cdk5/p35. LKE /XN001 also inhibits Cdk5/p25 ability to phosphorylate its target S522 residue in CRMP2 (collapsin response mediator protein-2) in a cell-free system. Furthermore, LKE/XN001 reduces Cdk5-targeted CRMP2 phosphorylation and neurofilament phosphorylation in the Cln3Δex7/8mouse modeling Batten disease (juvenile neuronal ceroid lipofuscinosis) while simultaneously reducing p25 levels in the Cln3Δex7/8mouse cerebellum. Molecular modeling identified a candidate LKE/XN001 binding site on the interface between the Cdk5 activation loop and its p25 binding partner. The putative binding interaction involves a network of hydrogen bonding interactions and a key salt bridge between LKE/XN001 and R156 of Cdk5, which could conceivably encourage a less productive conformation of the holoenzyme. Antagonism of Cdk5/p25 signaling pathways could explain the broad neuro-therapeutic profile of LKE/XN001 and provides a biological rational for translational development of LK analogs for neuropathologies. Disclosure KH is inventor of XN001 and holds equity in XoNovo Ltd.

Acute Hyperammonemia Induces NMDA-Mediated Hypophosphorylation of Intermediate Filaments Through PP1 and PP2B in Cerebral Cortex of Young Rats.

In the present work, we studied the effects of toxic ammonia levels on the cytoskeleton of neural cells, with emphasis in the homeostasis of the phosphorylating system associated with the intermediate filaments (IFs). We used in vivo and in vitro models of acute hyperammonemia in 10- and 21-day-old rats. In the in vivo model, animals were intraperitoneally injected with ammonium acetate (7mmol/Kg), and the phosphorylation level of the cytoskeletal proteins was analyzed in the cerebral cortex and hippocampus 30 and 60min after injection. The injected ammonia altered the IF phosphorylation of astrocytes (GFAP and vimentin) and neurons (neurofilament subunits of low, middle, and high molecular weight, respectively: NFL, NFM, and NFH) from cerebral cortex of 21-day-old rats. This was a transitory effect observed 30min after injection, recovering 30 min afterward. Phosphorylation was not altered in the cerebral cortex of 10-day-old pups. The homeostasis of hippocampal IFs was preserved at the studied ages and times. In the in vitro model, cortical slices of 10- and 21-day-old rats were incubated with 0.5, 1, or 5mM NH4Cl, and the phosphorylation level of the IF proteins was analyzed after 30min. The IF phosphorylation was not altered in cortical slices of 10-day-old rats; however, in cortical slices of 21-day-old pups, 5mM NH4Cl induced hypophosphorylation of GFAP and vimentin, preserving neurofilament phosphorylation levels. Hypophosphorylation was mediated by the protein phosphatases 1 (PP1) and 2B (PP2B), and this event was associated with Ca(2+) influx via N-methyl-D-aspartate (NMDA) glutamate receptors. The aim of this study is to show that acute ammonia toxicity targets the phosphorylating system of IFs in the cerebral cortex of rats in a developmentally regulated manner, and NMDA-mediated Ca(2+) signaling plays a central role in this mechanism. We propose that the disruption of cytoskeletal homeostasis could be an endpoint of the acute hyperammonemia in the developing brain. We believe that these results contribute for better understanding the molecular basis of the ammonia toxicity in brain.