Neurofilament Inclusions Research Articles

Striking intraneuronal neurofilament inclusions restricted to the locus coeruleus in a patient with Creutzfeldt-Jakob disease.

Striking intraneuronal neurofilament inclusions restricted to the locus coeruleus in a patient with Creutzfeldt-Jakob disease.

Striking intraneuronal neurofilament inclusions restricted to the locus coeruleus in a patient with Creutzfeldt-Jakob disease.

Striking intraneuronal neurofilament inclusions restricted to the locus coeruleus in a patient with Creutzfeldt-Jakob disease.

Abnormal neurofilament inclusions and segregations in dorsal root ganglia of a Charcot-Marie-Tooth type 2E mouse model.

Charcot-Marie-Tooth (CMT) disease or hereditary motor and sensory neuropathy is the most prevalent inherited peripheral neuropathy and is associated with over 90 causative genes. Mutations in neurofilament light polypeptide gene, NEFL cause CMT2E, an axonal form of CMT that results in abnormal structures and/or functions of peripheral axons in spinal cord motor neurons and dorsal root ganglion neurons. We have previously generated and characterized a knock-in mouse model of CMT2E with the N98S mutation in Nefl that presented with multiple inclusions in spinal cord neurons. In this report, we conduct immunofluorescence studies of cultured dorsal root ganglia (DRG) from NeflN98S/+ mice, and show that inclusions found in DRG neurites can occur in embryonic stages. Ultrastructural analyses reveal that the inclusions are disordered neurofilaments packed in high density, segregated from other organelles. Immunochemical studies show decreased NFL protein levels in DRG, cerebellum and spinal cord in NeflN98S/+ mice, and total NFL protein pool is shifted toward the triton-insoluble fraction. Our findings reveal the nature of the inclusions in NeflN98S/+ mice, provide useful information to understand mechanisms of CMT2E disease, and identify DRG from NeflN98S/+ mice as a useful cell line model for therapeutic discoveries.

Heterogeneity in the properties of NEFL mutants causing Charcot–Marie–Tooth disease results in differential effects on neurofilament assembly and susceptibility to intervention by the chaperone-inducer, celastrol

Aberrant aggregation of neurofilament proteins is a common feature of neurodegenerative diseases. For example, neurofilament light protein (NEFL) mutants causing Charcot–Marie–Tooth disease induce misassembly of neurofilaments. This study demonstrated that mutations in different functional domains of NEFL have different effects on filament assembly and susceptibility to interventions to restore function. The mouse NEFL mutants, NEFLQ333P and NEFLP8R, exhibited different assembly properties in SW13-cells, cells lacking endogenous intermediate filaments, indicating different consequences of these mutations on the biochemical properties of NEFL. The p.Q333P mutation caused reversible misfolding of the protein. NEFLQ333P could be refolded and form coil–coiled dimers, in vitro using chaotropic agent, and in cultured cells by induction of HSPA1 and HSPB1. Celastrol, an inducer of chaperone proteins, induced HSPA1 expression in motor neurons and prevented the formation of neurofilament inclusions and mitochondrial shortening induced by expression of NEFLQ333P, but not in sensory neurons. Conversely, celastrol had a protective effect against the toxicity of NEFLP8R, a mutant which is sensitive to HSBP1 but not HSPA1 chaperoning, only in large-sized sensory neurons, not in motor neurons. Importantly, sensory and motor neurons do not respond identically to celastrol and different chaperones are upregulated by the same treatment. Thus, effective therapy of CMT not only depends on the identity of the mutated gene, but the consequences of the specific mutation on the properties of the protein and the neuronal population targeted.

Role of proteasomes in the formation of neurofilamentous inclusions in spinal motor neurons of aluminum-treated rabbits

We examined the role of the 20S proteasome in pathologic changes, including abnormal aggregation of phosphorylated neurofilaments, of spinal motor nerve cells from aluminum-treated rabbits. Immunohistochemistry for the 20S proteasome revealed that many lumbar spinal motor neurons without intracytoplasmic neurofilamentous inclusions or with small inclusions were more intensely stained in aluminum-treated rabbits than in controls, whereas the immunoreactivity was greatly decreased in some enlarged neurons containing large neurofilamentous inclusions. Proteasome activity in whole spinal cord extracts was significantly increased in aluminum-treated rabbits compared with controls. Furthermore, Western blot analysis indicated that the 20S proteasome degraded non-phosphorylated high molecular weight neurofilament (neurofilament-H) protein in vitro. These results suggest that aluminum does not inhibit 20S proteasome activity, and the 20S proteasome degrades neurofilament-H protein. We propose that abnormal aggregation of phosphorylated neurofilaments is induced directly by aluminum, and is not induced by the proteasome inhibition in the aluminum-treated rabbits. Proteasome activation might be involved in intracellular proteolysis, especially in the earlier stages of motor neuron degeneration in aluminum-treated rabbits.

P1-049: Alzheimer-type neurofibrillary lesions and neurofilamentous inclusions are cell-type specific in transgenic mice expressing mutated tau proteins G272V/P301S

P1-049: Alzheimer-type neurofibrillary lesions and neurofilamentous inclusions are cell-type specific in transgenic mice expressing mutated tau proteins G272V/P301S

Neuropathological changes in ten cases of neuronal intermediate filament inclusion disease (NIFID): a study using α-internexin immunohistochemistry and principal components analysis (PCA)

Ten cases of neuronal intermediate filament inclusion disease (NIFID) were studied quantitatively. The alpha-internexin positive neurofilament inclusions (NI) were most abundant in the motor cortex and CA sectors of the hippocampus. The densities of the NI and the swollen achromatic neurons (SN) were similar in laminae II/III and V/VI but glial cell density was greater in V/VI. The density of the NI was positively correlated with the SN and the glial cells. Principal components analysis (PCA) suggested that PC1 was associated with variation in neuronal loss in the frontal/temporal lobes and PC2 with neuronal loss in the frontal lobe and NI density in the parahippocampal gyrus. The data suggest: 1) frontal and temporal lobe degeneration in NIFID is associated with the widespread formation of NI and SN, 2) NI and SN affect cortical laminae II/III and V/VI, 3) the NI and SN affect closely related neuronal populations, and 4) variations in neuronal loss and in the density of NI were the most important sources of pathological heterogeneity.

Laminar distribution of neurofilament inclusions and swollen achromatic neurons in neurofilament inclusion disease (NID)

AbstractThe laminar distribution of the neurofilament inclusions (NI) and swollen achromatic neurons (SN) was studied in gyri of the temporal cortex in four patients with neurofilament inclusion disease (NID). In 84% of gyri analysed, the density of the NI was maximal in the lower cortical laminae. The distribution of the SN was more variable than the NI. Density was maximal in the lower cortex in 46% of gyri, in the upper cortical laminae in 8% of gyri, and a bimodal distribution of 15% of gyri. In the remaining gyri, there was a more even distribution of SN with cortical depth. In 31% of gyri, the vertical density of the NI was positively correlated with that of the SN. The data suggest that cortical degeneration in the temporal lobe of NID initially affects neurons in the lower laminae. Subsequently, the pathology may spread to affect much of the cortical profile, the SN preceding the appearance of the NI.

Clustering of neuronal inclusions in "dementia with neurofilament inclusions".

Dementia with neurofilament inclusions (DNI) is a new disorder characterized clinically by early-onset dementia and histologically by the presence of intraneural inclusions immunopositive for neurofilament antigens but lacking tau and alpha-synuclein reactivity. We studied the clustering patterns of the neurofilament inclusions (NI) in regions of the temporal lobe in three cases of DNI to determine whether they have the same spatial patterns as inclusions in the tauopathies and alpha-synucleinopathies. The NI exhibited a clustered distribution (mean size of clusters 400 microm, range 50-800 microm, SD 687.8) in 24/28 of the areas studied. In 22 of these areas, the clusters exhibited a regular distribution along the tissue parallel to the pia mater or alveus. In 3 cortical areas, there was evidence of a more complex pattern in which the NI clusters were aggregated into larger superclusters. In 6 cortical areas, the size of the clusters approximated to those of the cells of origin of the cortico-cortical pathways but in the remaining areas cluster size was smaller than 400 microm. Despite the unique molecular profile of the NI, their spatial patterns are similar to those shown by filamentous neuronal inclusions in the tauopathies and alpha-synucleinopathies.

Patients with a novel neurofilamentopathy: dementia with neurofilament inclusions

We report a new disease, dementia with neurofilament inclusions, characterized clinically by early-onset dementia with frontal lobe signs, focal atrophy of the frontal and temporal lobes, and microscopically by the presence in many brain regions of intraneuronal, cytoplasmic, neurofilament inclusions. The neuronal inclusions are immunoreactive to all three molecular weight neurofilament subunits: heavy (NF-H), light, and medium subunits, including the phosphorylated and non-phosphorylated forms of NF-H. Prion protein and β-amyloid deposits were absent. The inclusions do not contain tau or α-synuclein protein aggregates known to characterize many neurodegenerative disorders. In addition to delineating a new disease entity, the identification of intraneuronal, cytoplasmic, neurofilament inclusions extends the molecular classification of neurodegenerative diseases and implicates new mechanisms of neurodegeneration in diseases affecting the human brain.

Deregulation of Cdk5 in a Mouse Model of ALS: Toxicity Alleviated by Perikaryal Neurofilament Inclusions

Recent studies suggest that increased activity of cyclin-dependent kinase 5 (Cdk5) may contribute to neuronal death and cytoskeletal abnormalities in Alzheimer's disease. We report here such deregulation of Cdk5 activity associated with the hyperphosphorylation of tau and neurofilament (NF) proteins in mice expressing a mutant superoxide dismutase ( SOD1 G37R ) linked to amyotrophic lateral sclerosis (ALS). A Cdk5 involvement in motor neuron degeneration is supported by our analysis of three SOD1 G37R mouse lines exhibiting perikaryal inclusions of NF proteins. Our results suggest that perikaryal accumulations of NF proteins in motor neurons may alleviate ALS pathogenesis by acting as a phosphorylation sink for Cdk5 activity, thereby reducing the detrimental hyperphosphorylation of tau and other neuronal substrates.

Motor neuron disease and trace element toxicity

There is a significant degree of overlap between the clinical syndromes of amyotrophic lateral sclerosis (ALS) and chronic AlCl3 neurotoxicity. Although there is little, if any, significant overlap with acute AlCl3 neurotoxicity, these latter studies are of importance in demonstrating that an exogenously administered neurotoxin can induce a posttranslational modification in neurofilament, sufficient to bring about neurofilamentous inclusions. The value in the study of chronic AlCl3 neurotoxicity is in the recapitulation of neuronal damage resembling that of ALS and the observations that suggest that the potential for recovery from the neuronal injury can be mediated by microglia. Studies are currently in progress to further define this potential. J. Trace Elem. Exp. Med. 13:131–140, 2000. © 2000 Wiley-Liss, Inc.

A knock on the head is not a good thing: head injury and Alzheimer’s disease-like pathology

Although a number of studies have suggested that head trauma increases the risk of developing Alzheimer’s disease (AD), other studies have not found such a link 1 Launer L.J. et al. Rates and risk factors for dementia and Alzheimer’s disease: results from EURODEM pooled analyses. EURODEM incidence research group and work groups. European studies of dementia. Neurology. 1999; 52: 78-84 Crossref PubMed Google Scholar . Two lines of evidence suggest that a link is likely. First, dementia pugilistica, or punch-drunk syndrome, is seen in ageing boxers and involves both motor symptoms (similar to those seen in Parkinson’s disease) and dementia. Second, several groups have reported that Alzheimer’s-like plaques and tangles are found in the brains of about one-third of patients who die soon after serious head trauma. The significance of these acute changes is unclear, and research has been slow because similar changes are not seen in rodent models. Smith et al.2 Smith D.H. et al. Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig. J. Neuropathol. Exp. Neurol. 1999; 58: 982-992 Crossref PubMed Scopus (215) Google Scholar used a pig model of brain injury to address this issue. The pig brain is gyrencephalic, possessing a structure with multiple folds, or sulci, more structurally similar to the human brain than are the rodent brains typically studied. The trauma used was mild, with all animals making a full recovery after 24 h. When their brains were examined one to ten days after trauma, about one-third of the animals had changes similar to the pathologic hallmarks of Alzheimer’s disease: accumulation of amyloid β-peptide (found in plaques in AD) and accumulation of abnormally phosphorylated tau protein (the major component of tangles in AD). However, unlike Alzheimer’s disease, most of the pathology was observed in injured axons rather than as extracellular plaques and tangles in gray matter. Some accumulation of hyperphosphorylated tau was seen in neurons, perhaps representing a ‘pre-tangle’, a precursor to the neurofibrillary tangle pathology seen in AD. Neuronal inclusions containing neurofilament proteins similar to those seen in Parkinson’s disease were also observed.

Accumulation of amyloid beta and tau and the formation of neurofilament inclusions following diffuse brain injury in the pig.

Brain trauma in humans increases the risk for developing Alzheimer disease (AD) and may induce the acute formation of AD-like plaques containing amyloid beta (A beta). To further explore the potential link between brain trauma and neurodegeneration, we conducted neuropathological studies using a pig model of diffuse brain injury. Brain injury was induced in anesthetized animals via nonimpact head rotational acceleration of 110 degrees over 20 ms in the coronal plane (n = 15 injured, n = 3 noninjured). At 1, 3, 7, and 10 days post-trauma, control and injured animals were euthanized and immunohistochemical analysis was performed on brain sections using antibodies specific for A beta, beta-amyloid precursor protein (betaPP), tau, and neurofilament (NF) proteins. In addition to diffuse axonal pathology, we detected accumulation of A beta and tau that colocalized with immunoreactive betaPP and NF in damaged axons throughout the white matter in all injured animals at 3-10 days post-trauma. In a subset of brain injured animals, diffuse A beta-containing plaque-like profiles were found in both the gray and white matter, and accumulations of tau and NF rich inclusions were observed in neuronal perikarya. These results show that this pig model of diffuse brain injury is characterized by accumulations of proteins that also form pathological aggregates in AD and related neurodegenerative diseases.

Overexpression of α-Internexin Causes Abnormal Neurofilamentous Accumulations and Motor Coordination Deficits in Transgenic Mice

alpha-Internexin is the first neuronal intermediate filament (IF) protein expressed in postmitotic neurons of the developing nervous system. In the adult, its expression is restricted to mature neurons in the CNS. To study the potential role of alpha-internexin in neurodegeneration, we have generated transgenic mice that overexpress rat alpha-internexin. The total levels of alpha-internexin expressed in the hemizygous and homozygous transgenic mice were approximately 2 and approximately 3 times the normal level, respectively. Overexpression of alpha-internexin resulted in the formation of cerebellar torpedoes as early as 1 month of age. These torpedoes are abnormal swellings of Purkinje cell axons that are usually seen in neurodegenerative diseases involving the cerebellum. EM studies showed accumulations of high levels of IFs and abnormal organelles in the torpedoes and soma of Purkinje cells, as well as in the large pyramidal neurons of the neocortex and in the ventral anterior and posteromedial nuclei of the thalamus. Behavioral tests demonstrate that these mice have a deficit in motor coordination as early as 3 months of age, consistent with the morphological neuronal changes. Our data further demonstrate that the neurofilamentous inclusions also lead to progressive loss of neurons in the aged transgenic mice. The motor coordination deficit and the loss of neurons are transgene dosage-dependent. These data yield direct evidence that high levels of misaccumulated neuronal IFs lead to neuronal dysfunction, progressive neurodegeneration, and ultimate loss of neurons. Moreover, the degrees of neuronal dysfunction and degeneration are proportional to the levels of misaccumulated neuronal IFs.

Time course of neuropathology in the spinal cord of G86R superoxide dismutase transgenic mice.

Transgenic mice with a G86R mutation in the mouse superoxide dismutase (SOD-1) gene, which corresponds to a mutation observed in familial amyotrophic lateral sclerosis (ALS), display progressive motor dysfunction leading to paralysis and premature death. In endstage SOD-1 transgenic mice, there is marked loss of spinal motor neurons and interneurons, accumulation of phosphorylated neurofilament inclusions, and reactive astrocytosis. The present study details the time course and ultrastructural appearance of these pathologic changes and correlates the timing of these events with the behavioral symptoms. There is no significant reduction in the number of total neurons, motor neurons, or interneurons in the ventral spinal cord of presymptomatic mice, as compared to age-matched control mice. In contrast, there is a significant reduction in the number of total neurons (-23.5%), motor neurons (-28.9%), and interneurons (-23.5%) in symptomatic SOD-1 transgenic mice. This neuron loss correlates temporally with the onset of reactive astrocytosis and the appearance of phosphorylated neurofilament inclusions. The identical timing of motor neuron and interneuron degeneration in this model of ALS strongly suggests that degeneration in the spinal cord of patients with ALS is not specifically directed at motor neurons, but rather more generally at several populations of neurons in the spinal cord. In addition, the late onset and rapid progression of neuron loss suggest that a toxic property is accumulating while the SOD-1 transgenic mice are presymptomatic, and that this toxic property must reach a threshold level before the onset of neuronal degeneration.

Neurofilaments and Orthograde Transport Are Reduced in Ventral Root Axons of Transgenic Mice that Express Human SOD1 with a G93A Mutation

Mice engineered to express a transgene encoding a human Cu/Zn superoxide dismutase (SOD1) with a Gly93 --> Ala (G93A) mutation found in patients who succumb to familial amyotrophic lateral sclerosis (FALS) develop a rapidly progressive and fatal motor neuron disease (MND) similar to amyotrophic lateral sclerosis (ALS). Hallmark ALS lesions such as fragmentation of the Golgi apparatus and neurofilament (NF)-rich inclusions in surviving spinal cord motor neurons as well as the selective degeneration of this population of neurons were also observed in these animals. Since the mechanism whereby mutations in SOD1 lead to MND remains enigmatic, we asked whether NF inclusions in motor neurons compromise axonal transport during the onset and progression of MND in a line of mice that contained approximately 30% fewer copies of the transgene than the original G93A (Gurney et al., 1994). The onset of MND was delayed in these mice compared to the original G93A mice, but they developed the same neuropathologic abnormalities seen in the original G93A mice, albeit at a later time point with fewer vacuoles and more NF inclusions. Quantitative Western blot analyses showed a progressive decrease in the level of NF proteins in the L5 ventral roots of G93A mice and a concomitant reduction in axon caliber with the onset of motor weakness. By approximately 200 d, both fast and slow axonal transports were impaired in the ventral roots of these mice coincidental with the appearance of NF inclusions and vacuoles in the axons and perikarya of vulnerable motor neurons. This is the first demonstration of impaired axonal transport in a mouse model of ALS, and we infer that similar impairments occur in authentic ALS. Based on the temporal correlation of these impairments with the onset of motor weakness and the appearance of NF inclusions and vacuoles in vulnerable motor neurons, the latter lesions may be the proximal cause of motor neuron dysfunction and degeneration in the G93A mice and in FALS patients with SOD1 mutations.

Aluminum inhibits neurofilament assembly, cytoskeletal incorporation, and axonal transport. Dynamic nature of aluminum-induced perikaryal neurofilament accumulations as revealed by subunit turnover.

The mechanism by which aluminum induces formation of perikaryal neurofilament (NF) inclusions remains unclear. Aluminum treatment inhibits: 1. The incorporation of newly synthesized NF subunits into Triton-insoluble cytoskeleton of axonal neurites; 2. Their degradation and dephosphorylation; 3. Their translocation into axonal neurites. It also fosters the accumulation of phosphorylated NFs within perikarya. In the present study, we addressed the relationship among these effects. Aluminum reduced the assembly of newly synthesized NF subunits into NFs. During examination of those subunits that did assemble in the presence of aluminum, it was revealed that aluminum also interfered with transport of newly assembled NFs into axonal neurites. Similarly, a delay in axonal transport of microinjected biotinylated NF-H was observed in aluminum-treated cells. Aluminum also inhibited the incorporation of newly synthesized and microinjected subunits into the Triton-insoluble cytoskeleton within both perikarya and neurites. Once incorporated into Triton-insoluble cytoskeletons, however, biotinylated subunits were retained within perikarya of aluminum-treated cells to a greater extent than within untreated cells. Notably, these subunits were depleted in the presence and absence of aluminum within 48 h, despite the persistence of the aluminum-induced perikaryal accumulation itself, suggesting that individual NF subunits undergo turnover even within aluminum-induced perikaryal accumulations. These findings demonstrate that aluminum interferes with multiple aspects of neurofilament dynamics and furthermore leaves open the possibility that aluminum-induced perikaryal NF whorls may not represent permanent structures, but rather may require continued recruitment of cytoskeletal constituents.

Spinal motor neuron neuroaxonal spheroids in chronic aluminum neurotoxicity contain phosphatase-resistant high molecular weight neurofilament (NFH)

It has previously been shown that a single intracisternal inoculum of AlCl 3 in young adult New Zealand white rabbits will induce a dose-dependent phosphatase resistance of the high molecular weight neurofilament protein (NFH) that is proportionate to the extent of neurofilamentous inclusion formation (Strong and Jakowec, 1994). To determine if the potential for dissolution of aluminum-induced neurofilamentous inclusions was dependant on the degree of NFH phosphatase resistance, we have examined NFH phosphatase sensitivity in a reversible chronic model of aluminum neurotoxicity. Rabbits receiving repeated intracisternal inoculums of 100 μg AlCl 3 at 28 day intervals until day 267 develop spinal motor neuron perikaryal and neuroaxonal neurofilamentous aggregates in a Stereotypic, dose-dependent fashion. In the rabbits receiving inoculums until day 156 with survival until day 267 without further aluminum exposure, neuroaxonal spheroids remained prominent while perikaryal inclusions largely resolved. Immunoreactivity to a monoclonal antibody recognizing phosphorylated NFH (SMI 31) was abolished in perikaryal aggregates at each time interval by dephosphorylation with bovine alkaline phosphatase. However, neuroaxonal spheroids maintained their immunoreactivity. Using time-course dephosphorylation studies of spinal cord homogenates, we observed a significant reduction in the rate of dephosphorylation of NFH following 267 days of AlCl 3 exposure ( P < 0.05). These observations suggest that neuroaxonal spheroids contain phosphatase-resistant NFH isoforms and that the potential for resolution of intraneuronal neurofilamentous inclusions correlates with the susceptibility of NF within these inclusions to enzymatic dephosphorylation.

Proximal sciatic axotomy does not inhibit the induction of neurofilamentous inclusions following intracisternal aluminum chloride exposure.

We have previously demonstrated an acute, dose-dependent suppression of low molecular weight neurofilaments (NFL) and intermediate molecular weight neurofilaments (NFM) steady state mRNA levels while sparing those of high molecular weight (NFH) mRNA 48 hours (h) following the intracisternal inoculation of AlCl3 in young adult New Zealand white rabbits. To determine whether this alteration in NF steady state mRNA stoichiometry is a necessary prerequisite to the induction of neurofilamentous inclusions, we examined the response of spinal motor neurons to aluminum exposure in vivo following axotomy. Forty-eight h following a complete transection of the proximal sciatic nerve, rabbits were inoculated intracisternally with either 1000 microg AlCl3 in 100 microl 0.9% NaCl or 0.9% NaCl alone. Rabbits were killed at either 48 or 120 h post-inoculation, and the extent of neurofilamentous inclusion formation quantified in both the cervical and the lumbosacral cord. Following the axotomy, rabbits developed an ipsilateral hind-limb paralysis. In spinal motor neurons ipsilateral to the axotomy, chromatolytic changes were observed and both NFH and NFM mRNA levels were significantly reduced (p<0.001). At 48 h post-AlCl3 inoculation, 29% of motor neurons contralateral to the axotomy demonstrated inclusions, whereas 43% of ipsilateral motor neurons demonstrated inclusions (Fisher's test, two tailed, p = 0.0196). At 120 h post-axotomy 75% and 83%, respectively, of neurons were involved (p = 0.0212). Neurofilamentous inclusions did not form in NaCl-inoculated rabbits. These observations indicate that an altered stoichiometry of NF mRNA steady levels, with a relative overexpression of NFH mRNA, is not critical to the induction of neurofilamentous inclusions following AlCl3 exposure.