NF-L), Medium Research Articles

SMA THERAPIES II AND BIOMARKERS: P.266Neurofilament as a potential biomarker for spinal muscular atrophy: rationale based on animal and human studies

SMA is an autosomal recessive neuromuscular disorder characterized by degeneration of alpha motor neurons (AHCs) in the spinal cord and brain stem leading to muscular atrophy and weakness. Prognostic and predictive biomarkers are needed to inform SMA treatment. Neurofilaments (NFs) have been proposed as biomarkers in disorders characterized by axonal degeneration, although their clinical utility has not been established. NFs are intermediate filaments uniquely expressed in neuronal cells and are integral components of the cytoskeleton. NFs are differentiated by molecular weight: light (NF-L), medium (NF-M), and heavy (NF-H). About 80% of axonal NFs are highly phosphorylated, conferring resistance to protein degradation. NFs are released into the extracellular fluid during axonal disintegration and are detected in cerebrospinal fluid (CSF) and blood. Advanced detection assays allow robust quantification of NF proteins in the CSF and blood of patients with amyotrophic lateral sclerosis and multiple sclerosis. End stage human SMA pathology is notable for loss of AHCs and ventral root axons, but the timing of degeneration based on disease severity is unknown. Severe SMA mouse models also demonstrate loss of AHCs and denervation of vulnerable muscle groups. In human SMA and mouse models, neuromuscular junctions (NMJs) are structurally immature and contain accumulations of pNFs at the presynaptic terminal, possibly from dysregulation of NF axonal transport with impaired NF degradation. Similar NF accumulations were observed in 70% of diaphragmatic NMJs in SMA Type I infants (age<6 months) versus normal controls. The restoration of NMJ synaptic integrity upon therapeutic restoration of survival motor neuron (SMN) in SMA mice suggests that sufficient SMN expression in the CNS prevents axonal degeneration and NF accumulation in SMA. Assessments of blood and CSF pNF-H levels are promising prognostic and predictive biomarkers for SMA disease activity and/or treatment efficacy.

Cortical Murine Neurons Lacking the Neurofilament Light Chain Protein Have an Attenuated Response to InjuryIn Vitro

Neurofilaments (NFs) have been proposed to have a significant role in attempted axonal regeneration following a variety of forms of injury. The NF triplet proteins of the central nervous system are comprised of light (NF-L), medium (NF-M) and heavy (NF-H) chains and are part of the type IV intermediate filament family. We sought to define the role of NF-L in the neuronal response to trauma and regeneration by examining the effect of total absence of the NF-L protein on neuronal maturation and response to axotomy. This study utilized an in vitro model comprising relatively mature cortical murine neurons derived from either wild-type embryonic (E15) mice or mice with a genetic knockout of NF-L (NF-L KO). Whilst NF-L KO neurons developed to relative maturity at a comparable rate to wild-type control neurons, NF-L KO neurons demonstrated relatively increased expression of α-internexin and decreased expression of NF-M. Further, we demonstrate that α-internexin co-immunoprecipitates with the NF binding protein NDel1 in NFL-KO cortical neurons in vitro. Following localized axotomy, NF-L KO neurons demonstrated reduced amyloid precursor protein accumulation in damaged neurites as well as a significant reduction in the number of axons regenerating (4.79+/-0.58 sprouts) in comparison to control preparations (10.47+/-1.11 sprouts) (p<0.05). These studies indicate that NFs comprising NF-L have a dynamic role in the reactive and regenerative changes in axons following injury.

Molecular Dynamics Study of Phosphorylation Mediated Structural Changes in Neurofilament Medium (NF-M) Subunit

Neurofilaments (NFs) are essential building blocks of axonal architecture. Abnormal behavior of these cytostructural elements has been associated with several neuromuscular disorders such as Amyotropic Lateral Sclerosis (ALS). NFs are assembled from three subunits: Low (NFL), Medium (NFM) and Heavy (NFH). These subunits are characterized by a common alpha helical rod domain and carboxyl terminal domains of different lengths specific to each subunit. The tails project from the core of the filament and contain a number of KSP repeat motifs that belongs to the sites for phosphorylation. Especially, the C-terminal tails of NFM and NFH that have relatively longer lengths and higher number of KSP repeats were found to be the key participants of the sidearm-mediated interfilament interactions that regulate the axonal diameter. Though it has been established that that the sidearms play a key functional role, little is known about the roles of individual subunits and the effect of phosphorylation on their behavior. Initially, it was believed that the NFH sidearms play a more integral role in determining axonal structure due to the presence of longer polypeptides and relatively higher KSP repeat units. However, recent studies showed that deleting NFH from neurofilaments does not affect axonal diameter, suggesting that NFM may in fact be the key player. In view of this, it is essential to have an understanding of the morphological behavior of the NFM sidearm in response to physiological conditions. In the present study we carried out MD simulations of human and mouse NFM C terminals under different phosphorylation and ionic conditions. The results from these studies provide useful molecular level insight into the structural changes of NFM sidearms in response to phosphorylation, ionic concentrations. The present study reveals sidearm-mediated regulation mechanism of axonal caliber.

After the storm

Neurofilaments (Nf), structural proteins of axons and dendrites, are composed of light (NfL), medium (NfM), and heavy (NfH) chain subunits. Due to their abundance and specificity for neurons, they have potential as markers of neuronal injury. Neuroaxonal damage releases Nf proteins into the extracellular space, CSF, and peripheral blood. Studies in diseases characterized by neuroaxonal loss (stroke, amyotrophic lateral sclerosis, Alzheimer disease, HIV-associated cognitive impairment, acute spinal cord injury, neuromyelitis optica, and multiple sclerosis [MS]) reported the utility of Nf as a potential surrogate marker. Nf studies in MS showed that levels correlated with disease activity. In 78% of patients with relapsing-remitting MS (RRMS) (but in no controls), there was detectable CSF NfL; levels rose after relapse and decreased over 3 months.1 A cross-sectional study reported detectable CSF NfL in 8% of controls, 48% of subjects with secondary progressive MS (SPMS), 44% with RRMS in remission, and 91% with RRMS who had relapsed in the preceding 3 months. Thirteen recent-relapse subjects studied longitudinally showed median NfL levels at baseline of 1.88 ng/mL, increasing to 2.70 ng/mL at 5 weeks and significantly raised at 2.02 ng/mL 15 weeks after relapse.2 A recent long-term study confirmed the utility of CSF NfL: baseline CSF NfL correlated with MS severity score (MSSS) with a median follow-up of 14 years.3 CSF NfL above the median conferred a higher risk for severe MS compared to subjects with a more benign course (odds …

Neurofilament localization and phosphorylation in the developing inner ear of the rat

Detailed understanding of neurofilament protein distribution in the inner ear can shed light on regulatory mechanisms involved in neuronal development of this tissue. We assessed the spatio-temporal changes in the distribution of neurofilaments in the developing rat inner ear between embryonic day 12 and 30 days after birth, using antibodies against phosphorylated as well as non-phosphorylated light (NFL), medium (NFM) and heavy (NFH) neurofilament subunits. Our results show that during development, the onset of neurofilament expression in the rat inner ear is on embryonic day 12, earlier than previously shown. We demonstrate that neurofilament subunits of different molecular weight emerge in a developmental stage-dependent order. In addition, we determined that neurofilaments of the vestibular nerve mature earlier than neurofilaments of the cochlear nerve. Cochlear neurofilament maturation progresses in a gradient from base to apex, and from inner to outer hair cells. The sequential pattern of neurofilament expression we describe may help understand the consequences of certain mutations, and contribute to develop therapeutic strategies.

Interplay between Liquid Crystalline and Isotropic Gels in Self-Assembled Neurofilament Networks

Neurofilaments (NFs) are a major constituent of nerve cell axons that assemble from three subunit proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight into a 10 nm diameter rod with radiating sidearms to form a bottle-brush-like structure. Here, we reassemble NFs in vitro from varying weight ratios of the subunit proteins, purified from bovine spinal cord, to form homopolymers of NF-L or filaments composed of NF-L and NF-M (NF-LM), NF-L and NF-H (NF-LH), or all three subunits (NF-LMH). At high protein concentrations, NFs align to form a nematic liquid crystalline gel with a well-defined spacing determined with synchrotron small angle x-ray scattering. Near physiological conditions (86 mM monovalent salt and pH 6.8), NF-LM networks with a high NF-M grafting density favor nematic ordering whereas filaments composed of NF-LH transition to an isotropic gel at low protein concentrations as a function of increasing mole fraction of NF-H subunits. The interfilament distance decreases with NF-M grafting density, opposite the trend seen with NF-LH networks. This suggests a competition between the more attractive NF-M sidearms, forming a compact aligned nematic gel, and the repulsive NF-H sidearms, favoring a more expansive isotropic gel, at 86 mM monovalent salt. These interactions are highly salt dependent and the nematic gel phase is stabilized with increasing monovalent salt.

The calpain inhibitor MDL‐28170 and the AMPA/KA receptor antagonist CNQX inhibit neurofilament degradation and enhance neuronal survival in kainic acid‐treated hippocampal slice cultures

The cytoskeleton controls the architecture and survival of the central nervous system neurons by maintaining the stability of axons, dendrites and cellular architecture, and any disturbance in this genuine structure could compromise cell survival. The developmentally regulated intracellular intermediate filament protein neurofilament (NF), composed of the light (NF-L), medium (NF-M) and high (NF-H) molecular weight isoforms, is expressed abundantly in nerve cells but its significance in nerve cell survival in stress situations in the brain is unknown. We have used Western blotting, immunocytochemistry, and Fluoro-Jade B and thionine stainings to clarify the effect of kainic acid (KA) treatment on NF protein stability, and its importance for neuronal survival in hippocampal slice cultures. The contribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/KA glutamate receptor subtypes, calpain proteases and L-type Ca2+-channels to these processes were also assessed. Our results indicated that KA-induced degradation of NF was a fast process, similarly affecting all three NF proteins. It was effectively inhibited by the AMPA/KA receptor antagonist CNQX and the calpain inhibitor MDL-28170, whereas the Ca2+-channel blocker nifedipine and the NMDA receptor antagonist MK-801 had no significant effect. Moreover, KA-induced neuronal damage was effectively decreased in cultures treated with CNQX and MDL-28170. Our results suggest that the stability of NF proteins is an important factor contributing to neuronal survival after excitotoxic injury, and that both AMPA/KA receptor antagonists and calpain inhibitors might serve as neuroprotectants against this type of insult in the immature hippocampus.

Timing of Neuronal Intermediate Filament Proteins Expression in the Mouse Vomeronasal Organ During Pre- and Postnatal Development. An Immunohistochemical Study

Several types of intermediate filament proteins are expressed in developing and mature neurons; they cooperate with other cytoskeletal components to sustain neuronal function from early neurogenesis onward. In this work the timing of expression of nestin, peripherin, internexin, and the neuronal intermediate filament triplet [polypeptide subunits of low (NF-L), medium (NF-M), and high (NF-H) molecular weight] was investigated in the developing fetal and postnatal mouse vomeronasal organ (VNO) by means of immunohistochemistry. The results show that the sequence of expression of intermediate filament proteins is internexin, nestin, and NF-M in the developing vomeronasal sensory epithelium; internexin, peripherin, and NF-M in the developing vomeronasal nerve; and nestin, internexin and peripherin, NF-L, and NF-M in the nerve supply to accessory structures of the VNO. At sexual maturity (2 months) NF-M is only expressed in vomeronasal neurons and NF-M, NF-L and peripherin are expressed in extrinsic nerves supplying VNO structures. The differential distribution of intermediate filament proteins in the vomeronasal sensory epithelium and nerve is discussed in terms of the cell types present therein. It is concluded that several intermediate filament proteins are sequentially expressed during intrauterine development of the VNO neural structures in a different pattern according to the different components of the VNO.

Quantitative evidence for neurofilament heavy subunit aggregation in motor neurons of spinal cords of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease of unknown etiology, affects motor neurons leading to atrophy of skeletal muscles, paralysis and death. There is evidence for the accumulation of neurofilaments (NF) in motor neurons of the spinal cord in ALS cases. NF are major structural elements of the neuronal cytoskeleton. They play an important role in cell architecture and differentiation and in the determination and maintenance of fiber caliber. They are composed of three different polypeptides: light (NF-L), medium (NF-M) and heavy (NF-H) subunits. In the present study, we performed a morphological and quantitative immunohistochemical analysis to evaluate the accumulation of NF and the presence of each subunit in control and ALS cases. Spinal cords from patients without neurological disease and from ALS patients were obtained at autopsy. In all ALS cases there was a marked loss of motor neurons, besides atrophic neurons and preserved neurons with cytoplasmic inclusions, and extensive gliosis. In control cases, the immunoreaction in the cytoplasm of neurons was weak for phosphorylated NF-H, strong for NF-M and weak for NF-L. In ALS cases, anterior horn neurons showed intense immunoreactivity in focal regions of neuronal perikarya for all subunits, although the difference in the integrated optical density was statistically significant only for NF-H. Furthermore, we also observed dilated axons (spheroids), which were immunopositive for NF-H but negative for NF-M and NF-L. In conclusion, we present qualitative and quantitative evidence of NF-H subunit accumulation in neuronal perikarya and spheroids, which suggests a possible role of this subunit in the pathogenesis of ALS.

Changes in neurofilament protein-immunoreactivity after kainic acid treatment of organotypic hippocampal slice cultures.

Neurofilament (NF) proteins are expressed in the majority of neurons in the central nervous system, and play a crucial role in the organization of neuronal shape and function. In the present study, we have used immunoblotting and immunocytochemical methods to study the light (NF-L), medium (NF-M ), and heavy (NF-H) molecular weight NF proteins in cultured organotypic hippocampal slices during the in vitro maturation and the changes after kainic acid (KA) treatment. In control cultures at 11 DIV throughout 25 DIV, CA3 pyramidal neurons and their proximal dendrites were heavily labeled with the antibodies against all three NF proteins. In CA1 pyramidal neurons, no staining was detected in any age group. A few weakly NF-L positive granule cells with fibers were detected in each age group, whereas NF-M and NF-H positive granule cells first appeared in the older cultures. The application of KA (5 microM) to the cultures for 48 hr, induced a pronounced cell death in the CA3 cell layers, and also moderately damaged granule cells. After the treatment, the immunoblot signal of NF-L and NF-M markedly decreased, whereas that of NF-H almost completely disappeared. The amount of NF-L positive fibers, however, dramatically increased in the molecular and hilar regions of the dentate gyrus in both age groups. Our results show the cellular heterogeneity in the distribution of NF protein triplet in cultured organotypic hippocampal slices. Kainic acid treatment induced changes, which mimicked those observed in the hippocampal region of epileptic animals.

Transcription of intermediate filament genes is enhanced in focal cortical dysplasia.

Focal cortical dysplasia (FCD) is characterized by disorganized cerebral cortical cytoarchitecture. Increased expression of several intermediate filament (IF) proteins such as neurofilament, vimentin, alpha-internexin, and nestin observed in dysplastic "balloon" neurons (DN) may contribute to disrupted cortical lamination. We hypothesized that increased IF protein expression results from enhanced IF gene transcription within dysplastic neurons. We used a novel strategy to evaluate IF mRNA expression in three FCD specimens from medically intractable epilepsy patients. Poly(A) mRNA was amplified (aRNA) from single microdissected DN, morphologically normal neurons at the margin of the FCD resection, morphologically normal neurons in non-FCD cortex from epilepsy patients, and normal control neurons. Radiolabeled aRNA from single neurons was used to probe cDNA arrays containing the low (NFL), medium (NFM) and high (NFH) molecular weight neurofilament isoform, alpha-internexin, desmin, vimentin, peripherin (PRPH), nestin, and glial fibrillary acidic protein (GFAP) cDNAs. Hybridization intensity of aRNA-cDNA hybrids was used to quantify relative IF abundance. Increased expression of nestin, alpha-internexin, PRPH, vimentin, NFL, NFM, and NFH mRNAs was found in DN when compared with the three control neuronal subtypes. Desmin and GFAP mRNAs were not detected in any cell types. Expression of PRPH mRNA and protein in select DN was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry. We conclude that aberrant expression of IF proteins in FCD likely results from enhanced transcription of IF genes in dysplastic neurons and propose that future analysis of transcriptional elements that regulate IF expression be evaluated in FCD.

Expression of neurofilament gene in spinal motoneurons during neural regeneration

The techniquein situ hybridization was used to measure the levels of light (NF-L), medium (NF-M) and heavy (NF-H) neurofilament protein subunits mRNA in L4-6 spinal motoneurons in adult rat during regeneration following a unilateral crush of the sciatic nerve. It was found that the hybridization signals of each neurofilament subunit rnRNA were dramatically decreased in spinal motoneurons postaxotomy by light microscopy. The hybridization signals of NF-L and NF-M mRNA were located in cytoplasm of neurons, whereas NF-H mRNA was found in both nucleus and cytoplasm of neurons. Image analysis showed that the encoding levels of mRNA for each of neurofilament subunit mRNA reduced on the 3rd d and returned to control levels on the 28th d following the lesion. The relative levels of mRNA coding for each neurofilament subunit were significantly different. The lowest level of NF-L mRNA was observed at 5 d postaxotomy, and that of NF-M, NF-H mRNA on the 7th and 10th d after injury. Moreover, the levels of HF-M and NF-H mRNA were reduced much lower and lasted much longer than that of NF-L mRNA. The observations suggest that there were different mechanisms for the regulation of neurofilament subunit genes expression. The reduced neurofilament gene expression may be due to a response to axonal injury and advance the restructure of axonal architecture.