A systematic review and meta-analysis of CSF neurofilament protein levels as biomarkers in dementia.

BACKGROUND: Loss of cortical neurons is a key pathological feature in neurodegenerative dementias. Cerebrospinal fluid (CSF) neurofilaments (Nf) are a biomarker for neuronal death and axonal loss. OBJECTIVE: To perform a meta-analysis to investigate the value of CSF Nf levels for the laboratory-supported differential diagnosis of neurodegenerative dementias. METHODS: A systematic review and meta-analysis of studies on CSF Nf heavy (NfH) and light (NfL) levels in patients with dementia. The dementia subgroups analysed were Alzheimer (AD), frontotemporal lobe dementia (FTLD), vascular dementia (SVD), minimal cognitive deficit (MCI). RESULTS: We identified 12 studies on CSF NfH and NfL levels which met the inclusion criteria and 11 were of a quality good enough to be used in this meta-analysis. CSF data was available on 818 patients (306 AD, 106 SVD, 98 FTLD, 25 MCI, 283 controls). Overall CSF NfH and NfL levels were higher in patients with AD, FTLD and SVD when compared to controls. The size of the effect ranged from 0.71 to 1.38. The strongest effect was observed for the comparison of FTLD patients with controls, both for NfL (1.38) and NfH (0.74). CSF NfL were also able to separate patients with FTLD from those with AD. CONCLUSION: At present we cannot recommend CSF NfH and NfL levels for use as a screening test in the diagnosis of dementia because of the rather small effect size. However, both neurofilament proteins may be of value for targeted investigation of some patients with FTLD, SVD and AD.

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin.

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.

Proteasome inhibition induces selective motor neuron death in organotypic slice cultures.

A dysfunctional ubiquitin-proteasome system recently has been proposed to play a role in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We have shown previously that spinal motor neurons are more vulnerable to proteasome inhibition-induced neurotoxicity, using a dissociated culture system. To confirm this toxicity, we used organotypic slice cultures from rat neonatal spinal cords, which conserve the structure of the spinal cord in a horizontal plane, enabling us to identify motor neurons more accurately than in dissociated cultures. Furthermore, such easy identifications make it possible to follow up the course of the degeneration of motor neurons. When a specific proteasome inhibitor, lactacystin (5 microM), was applied to slice cultures, proteasome activity of a whole slice was suppressed below 30% of control. Motor neurons were selectively damaged, especially in neurites, with the increase of phosphorylated neurofilaments. They were eventually lost in a dose-dependent manner (1 microM, P < 0.05; 5 microM, P < 0.01). The low capacity of Ca(2+) buffering is believed to be one of the factors of selectivity for damaged motor neurons in ALS. In our system, negative staining of Ca(2+)-binding proteins supported this notion. An intracellular Ca(2+) chelator, BAPTA-AM (10 microM), exerted a significant protective effect when it was applied with lactacystin simultaneously (P < 0.01). We postulate that proteasome inhibition is an excellent model for studying the mechanisms underlying selective motor neuron death and searching for new therapeutic strategies in the treatment of ALS.

Glutamate induces rapid loss of axonal neurofilament proteins from cortical neurons in vitro.

One of the primary hallmarks of glutamate excitotoxicity is degradation of the neuronal cytoskeleton. Using a tissue culture approach, we have investigated the relationship between excitotoxicity and cytoskeletal degradation within axons, with particular reference to the axon specific neurofilament proteins. Neurofilaments were rapidly lost from axons over a 24-h period in response to excitotoxic insult (as observed by immunocytochemistry and western blotting), while other axonal cytoskeletal markers (such as betaIII-tubulin) remained intact. Treatment with kainic acid and NMDA, or complementary experiments using the pharmacological glutamate receptors blockers CNQX (kainate/AMPA receptor antagonist) and MK-801 (NMDA receptor antagonist), demonstrated that neurofilament degeneration was mediated primarily by NMDA receptor activity. This work suggests that excitotoxicity triggers a progressive pathway of cytoskeletal degeneration within axons, initially characterised by the loss of neurofilament proteins.

Lamprey neurofilaments contain a previously unreported 50-kDa protein.

We have previously hypothesized that regeneration of axons after spinal cord injury in the lamprey may involve assembly and transport of neurofilaments (NFs) into the growing tip. A single NF, NF-180, has been cloned in this laboratory and until now was thought to be the only NF subunit in lamprey nervous system. However, homopolymerization of NF-180 has not been observed either in experiments on transfected cells or in self-assembly tests in vitro. Forty-three monoclonal antibodies designated as LCM series were generated previously against cytoskeletal proteins of the lamprey nervous system. Seven LCMs were NF specific, and five were keratin specific, as demonstrated by immunohistochemistry. In the present study, one antibody, LCM40, selectively labeled axons in immunohistochemical sections and recognized a single 50-kDa protein in Western blots. Other neuron-specific LCMs and anti-NF antibodies, e.g., LCM39, recognized a known NF subunit, NF-180. Two-dimensional (2-D) gel electrophoresis was employed to separate otherwise indistinguishable individual cytoskeletal proteins. Western blot analysis with an antibody (IFA) that selectively labels all known intermediate filaments indicated that this 50-kDa protein is an intermediate filament (IF). The new protein was incorporated into IF polymers in vitro. Immunoelectron microscopy confirmed that neuronal IFs contain this novel protein. These results suggest that the 50-kDa protein is a previously unrecognized neuronal IF subunit in the lamprey.

A specific ELISA for measuring neurofilament heavy chain phosphoforms.

Neurofilaments (Nf) are the major constitutents of the axoskeleton and body fluid Nf levels are an important tool for estimating axonal degeneration in vivo. This paper presents a new sandwich ELISA allowing quantification of the NfH(SMI35) phosphoform from CSF, brain tissue and cell culture homogenates. The sensitivity of the NfH(SMI35) ELISA is 0.2 ng/ml with a recovery of 119% and a mean within- and between-batch precision of 10.6% and 23%, respectively. CSF NfH(SMI35) was stable at 4 degrees C, is not influenced by freeze-thaw cycles, and proteolysis present at room temperature could be prevented by adding protease inhibitors. Aggregate formation was observed for HPLC-purified bovine NfH and could be resolved by sonication. The upper reference value for CSF NfH(SMI35) levels (0.73 ng/ml) was defined as the 95% cumulative frequency from 416 CSF samples. Based on this cutoff, a significantly higher proportion of patients with amyotrophic lateral sclerosis, space-occupying lesions, disc prolapse and subarachnoid haemorrhage had pathologically elevated NfH(SMI35) levels compared to patients with cluster headache or demyelinating disease.A new nomenclature is proposed to facilitate the comparison between ELISA, immunoblotting and immunocytochemistry.

The correspondence between the labeling patterns of antibody RT97, neurofilaments, microtubule associated protein 1B and tau varies with cell types and development stages of chicken retina.

The correspondence between the labeling patterns of antibody RT97, neurofilaments (NF-M and NF-H), microtubule associated protein 1B (MAP1B) and tau, were studied in the developing chicken. At embryonic day 3 (E3), intense RT97 immunoreactivity (IR) was found to be localized in cells in the region adjacent to the intraretinal space, which separates the inner and outer layers of the optic cup, and this was sustained at E8. However, this pattern changed dramatically at E12, as the intensities of RT97 IR increased in the inner retinal layer, while the outermost layers showed only weak IR. The adult stage retina showed RT97 IR within the nerve fibers of the ganglion cells, the processes of the amacrine cells and the photoreceptors. Additional immunostainings for NF-M, -H, MAP1B and tau showed that the observed changes in RT97 IRs were due to the different expressions of these proteins at different development stages.

Neurofilament expression in the rat brain after cerebral infarction: effect of age.

In this study the role of neurofilaments (NFs) in brain plasticity after cerebral infarction in young and middle aged rats was evaluated. Focal cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery in 3- and 20-month-old male Sprague-Dawley rats. After 1 week, brains were removed and in situ hybridization and immunostaining was performed for NF-68 kDa, 160 kDa and 200 kDa in different phosphorylation states. After focal cerebral ischemia the levels of gene and protein expression of neurofilament proteins were increased in the border zone of the infarcted area compared with the unaffected contralateral site. Furthermore, the level of gene expression was significant lower in aged as in young animals. Focal cerebral ischemia resulted in a clearly increased number of immunostained axons in the penumbral region in both young and aged rats. On the other hand the immunostained apical dendrites became thicker and vacuolization appeared. Our results suggest that that neurofilament proteins are involved in response of brain to focal ischemia.

Polyreactive autoimmune response induced by PC 12 cell grafts into rat striatum.

In order to better characterize the autoantibodies induced by PC12 cells grafted into rat brain, we have tested sera from these animals by immunoblotting with several preparations, including phosphorylated and dephosphorylated neurofilaments, keratins, PC12 cells and proteins from various rat tissues, and by immunofluorescence of rat spinal cord neurons in culture. Sera from grafted rats reacted with several antigens present in all tissues tested and stained in cultured neurons not only NF but also cell bodies and membranous granular structures. These observations suggest either the polyreactivity of autoantibodies or the induction of a polyclonal B cell activation consecutive to the release of central nervous system antigens into the blood stream. These results are discussed with regard to the role of NF autoantibodies in neurodegenerative diseases.