Bone marrow stromal cell transplantation through tail vein injection promotes angiogenesis and vascular endothelial growth factor expression in cerebral infarct area in rats.

BACKGROUND AIMS: This study sought to identify correlations between vascular endothelial growth factor (VEGF) expression after tail-vein injection of rat-derived bone marrow stromal cells (BMSCs) and neurogenesis and angiogenesis in cerebral infarct of rats. METHODS: Rats with intraluminal middle cerebral artery occlusion were injected in a tail vein with Hoechst-labeled BMSCs. Functional recovery from cerebral infarction was observed through the use of a locomotion score. The brains of injected rats were sliced, and Hoechst-labeled BMSCs in the infarct and peri-infarct areas and subventricular zone (SVZ) were detected with the use of fluorescence microscopy. Ki-67 (as a cell proliferation marker) and VEGF expression were determined by means of immunohistochemistry. Neurofibril formation and angiogenesis were examined by means of Bielschowsky staining. RESULTS: Within 1 to 2 weeks after BMSC injection, rats showed significantly improved locomotion scores compared with rats without BMSC injection (P < 0.01). Viable BMSCs were found in the peri-infarct area. The numbers of Ki-67-positive and VEGF-positive cells in the peri-infarct area and SVZ of injected rats were significantly increased compared with the control group (P < 0.01). Numerous new vessels, neurofibrils and anastomosed vasculatures were present in the infarct area. These neurofibrils mainly surrounded the neovasculatures. CONCLUSIONS: These results indicate that BMSC-transplantation in rats through tail vein injection can increase neurogenesis, perhaps as the result of VEGF-mediated and/or Ki-67-mediated angiogenesis.

Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport.

OBJECTIVE: The lesions of Parkinson disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. METHODS: We grew primary cortical mouse neurons in microfluidic devices to separate somata from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. RESULTS: Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. INTERPRETATION: These results support the hypothesis that the progression of Parkinson disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be trans-synaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extracellular phase of their journey.

Cajal's second great battle for the neuron doctrine: the nature and function of neurofibrils.

One hundred years ago, a novel kind of reticularism threatened to displace the neuron doctrine as the established model of functional organization of the nervous system. The challenging paradigm, championed by Stephan von Apáthy and Albrecht Bethe, held that nerve impulses propagate along neurofibrils connected in a continuous network throughout all nerve cells. Santiago Ramón y Cajal, a leading figure in the conception of the neuron doctrine, headed again the battle against this return of reticularism. Dissatisfied with the available staining techniques, he devised the "reduced silver nitrate method" that even Camillo Golgi recognized as the best at the time for revealing the neurofibrils. In 1904 Cajal already published over a dozen papers in three languages describing neurofibril distributions in the nervous systems of diverse vertebrates and invertebrates, under both normal and experimental conditions. Next he investigated the involvement of neurofibrils in the process of nerve regeneration. This unprecedented survey led him to the conclusion that the neurofibrils are linear "colonies" of particles constituting a semi-solid, dynamic internal skeleton of the nerve cell. Apáthy reacted with a long invective paper that Cajal had no choice but acknowledging. His comprehensive reply, published in 1908, meant the effective end of the renewed reticularist campaign against the neuron doctrine. Along the way, a visionary and today almost forgotten chapter in the history of the cytoskeleton had also been written.

Centrifugal effects in the avian retina.

Electrical stimulation of the centrifugal fibers to the avian retina can disturb the balance between the excitatory and inhibitory system within the receptive fields of individual retinal ganglion cells. Although the mechanisms may vary from one unit to another, the effect is always to make them fire more readily and to a wider range of visual inputs.

Ciliary orientation: controlled by cell membrane or by intracellular fibrils?

The cirri of the ciliate Euplotes all asslumne the "reversed" orientation whenever the cell is depolarized and the "forward" orientation whenever the cell is hyperpolarized. Potenitial changes arise spontaneolusly or are induced by electrical or mechanical stimuli. The orientation responses of thte cirri are appatently independent of intracellular "neuromotor" fibrils previously assigned a coordinating function, as they persist after the fibrils are transected.

Recent advances in experimental modeling of the assembly of tau filaments.

Intracellular assembly of microtubule-associated protein tau into filamentous inclusions is central to Alzheimer's disease and related disorders collectively known as tauopathies. Although tau mutations, posttranslational modifications and degradations have been the focus of investigations, the mechanism of tau fibrillogenesis in vivo still remains elusive. Different strategies have been undertaken to generate animal and cellular models for tauopathies. Some are used to study the molecular events leading to the assembly and accumulation of tau filaments, and others to identify potential therapeutic agents that are capable of impeding tauopathy. This review highlights the latest developments in new models and how their utility improves our understanding of the sequence of events leading to human tauopathy.

Neurofilaments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites.

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

Neurofilaments are nonessential to the pathogenesis of toxicant-induced axonal degeneration.

Axonal neurofilament (NF) accumulations occur before development of symptoms and before other pathological changes among idiopathic neurodegenerative diseases and toxic neuropathies, suggesting a cause-effect relationship. The dependence of symptoms and axonal degeneration on neurofilament accumulation has been tested here in a transgenic mouse model (Eyer and Peterson, 1994) lacking axonal NFs and using two prototypic toxicant models. Chronic acrylamide (ACR) or 2,5-hexanedione exposure resulted in progressive and cumulative increases in sensorimotor deficits. Neurobehavioral tests demonstrated similar expression of neurotoxicity in transgenic (T) mice and their nontransgenic (NT) littermates (containing normal numbers of axonal NFs). Axonal lesions were frequently observed after exposure to either toxicant. Quantitation of ACR-induced lesions demonstrated the distal location of pathology and equal susceptibility of T and NT axons. We conclude that axonal NFs have no effect on neurotoxicity and the pattern of pathology in these mammalian toxic neuropathies. These results also suggest that the role of neurofilament accumulation in the pathogenesis of neurodegenerative diseases requires careful evaluation.

Excitable neurofibrils and the problem of identifying the structure of central excitatory synapses in the nineteenth century.

Neurofibrils, identified after staining with Cajal's reduced silver nitrate, for example, were thought by many senior histologists in the nineteenth and early-twentieth centuries to conduct action potentials. There was no basis for this popular idea, although it was the impetus for intense study of the "neurofibrillar network" within neurons by Golgi, Cajal, Freud, and many others. Here, I trace the way in which this "excitable neurofibrillary" hypothesis led to major problems in the attempt by histologists to identify the central excitatory synapse, postulated by Sherrington on functional grounds and eventually described by Berkley.

Mechanism of neurofibrillary degeneration in Alzheimer's disease.

Neurofibrillary degeneration associated with the formation of intraneuronal neurofibrillary tangles of paired helical filaments (PHF) and 2.1 nm tau filaments is one of the most characteristic brain lesions of Alzheimer's disease. The major polypeptides of PHF are the microtubule associated protein tau. tau in PHF is present in abnormally phosphorylated forms. In addition to the PHF, the abnormal tau is present in soluble non-PHF form in the Alzheimer's disease brain. The level of tau in Alzheimer's disease neocortex is severalfold higher than in aged control brain, and this increase is in the form of the abnormally phosphorylated protein. The abnormally phosphorylated tau does not promote the assembly of tubulin into microtubules in vitro, and it inhibits the normal tau-stimulated microtubule assembly. After in vitro dephosphorylation both PHF and non-PHF abnormal tau stimulate the assembly of tubulin into microtubules. The activities of phosphoseryl/phosphothreonyl protein phosphatase 2A and nonreceptor phosphotyrosyl phosphatase(s) are decreased in AD brain. It is suggested that 1. A defect(s) in the protein phosphorylation/dephosphorylation system is one of the early events in the neurofibrillary pathology in AD; 2. A decrease in protein phosphatase activities, at least in part, allows the hyperphosphorylation of tau; and 3. Abnormal phosphorylation and polymerization of tau into PHF most probably lead to a breakdown of the microtubule system and consequently to neuronal degeneration.

Abnormal tau species are produced during Alzheimer's disease neurodegenerating process.

Tau proteins were detected in human brain using two polyclonal antibodies: anti-paired helical filaments and anti-human native tau proteins. Both antisera detected identically the normal set of tau proteins in control brains. Moreover they detected two abnormal tau variants of 64 and 69 kDa exclusively in brain areas showing neurofibrillary tangles and senile plaques. Tau 64 and 69 were abnormally phosphorylated as revealed by the decrease in their molecular mass observed after alkaline phosphatase treatment. Therefore, tau 64 and 69 are specific markers of the neurofibrillary degeneration of the Alzheimer type and might be useful tools for studying the first pathological events that lead to neuronal death.

Alzheimer's disease presenilin-1 expression modulates the assembly of neurofilaments.

Mutations in presenilin-1 gene are responsible for the majority of early-onset familial Alzheimer's disease cases. The function of this protein and the mechanism underlying the pathogenicity of its mutations are still unclear. To elucidate the role of presenilin-1 in the Alzheimer's disease pathology, we tested two such mutations (P117L and M146L) for their effect in stably transfected mouse neuroblastoma cell lines. Over-expression of the wild-type presenilin-1 gene induced formation of a well-extended, orderly organized network consisting of neurofilaments assembled from the L and H subunits, while in cells with the mutant gene this network was markedly reduced to short filaments concentrated in structures resembling cups. Cells expressing the mutant gene displayed altered processing of the transgene protein and neurofilament-H, suggesting that presenilin-1 is the mediator of changes targeted at neurofilaments. The two different mutations produced similar alterations, implying that this is a common pathogenic mechanism. Presenilin-1, neurofilament-H and tau proteins showed co-localization as evidenced by confocal microscopy, suggesting a possible physiological connection between these three proteins. Presenilin-1 appears to influence assembly of the H subunit into neurofilaments and the subsequent formation of new neurites. Mutations impair this function of presenilin-1, resulting in inhibition of neurite outgrowth. That presenilin-1 influences the assembly of neurofilaments may represent a novel pathway through which presenilin-1 mutations are involved in Alzheimer's disease pathology. In this hypothesis, presenilin-1 mutations will be associated with aberrant sprouting leading to synaptic loss, a key neuropathological feature of Alzheimer's disease.

ApoE and Abeta1-42 interactions: effects of isoform and conformation on structure and function.

Abnormalities in the processing of amyloid precursor protein to amyloid-beta (Abeta) are causal factors, and the presence of the epsilon4 allele of apolipoprotein E (apoE) is the primary risk factor for Alzheimer's disease (AD). Based, at least in part, on these genetics, the potential structural and functional interactions between these two proteins are the focus of our research. To understand the nature of the physical interactions between apoE and Abeta, we initially utilized gel-shift assays to demonstrate that native apoE2 and E3 (associated with lipid particles) form an SDS-stable complex with Abeta that is more abundant than the apoE4:Abeta complex. We further demonstrated that exogenous apoE3 but not E4 prevents Abeta-induced neurotoxicity by a process that requires apoE receptors. In addition, both exogenous apoE3 and E4 prevent Abeta-induced, glial-mediated inflammation, also via a process that requires apoE receptors. These functional effects all occur at a molar ratio of apoE to Abeta of 1:30. Because the biological activities for both apoE and Abeta are profoundly influenced by their isoform and conformation, respectively, we further investigated the idea that apoE3 and E4 differentially interact with particular aggregation species of Abeta1-42. Our overall hypothesis is that apoE has two general functions in relation to Abeta. First, apoE interacts with oligomeric Abeta via an apoE receptor-mediated process to inhibit neurotoxicity and neuroinflammation (apoE3 > apoE4) a process possibly related to binding and clearance of apoE3:oligomer complexes. Second, apoE facilitates the deposition of Abeta as amyloid (apoE4 > apoE3). We will continue to investigate the effect of apoE isoform and Abeta conformation on the structural and functional interactions between these two proteins in relation to the pathogenesis of AD.

Aluminum-induced neurofibrillary degeneration disrupts acquisition of the rabbit's classically conditioned nictitating membrane response.

Rabbits received intraventricular injections of aluminum chloride, hydrochloric acid, or served as unoperated controls. On the 6th day postsurgery, they underwent 4 days (100 trials per day) of classical conditioning of the nictitating membrane response (NMR) to a tone conditioned stimulus and an air-puff unconditioned stimulus. Unoperated and hydrochloric acid control animals readily acquired the conditioned response. Aluminum intoxicated rabbits, in contrast, did not acquire the conditioned response over the 4 days of testing. This disruption of conditioning in aluminum-treated rabbits could not be attributed to deficits in sensory or motor processes or to illness. Neuropathological analysis revealed widespread neurofibrillary tangle formation in aluminum-treated animals. Furthermore, the degree of neurofibrillary degeneration was significantly negatively correlated with the degree of conditioning. The results are considered in the context of using the rabbit NMR preparation as a model system for studying age-related conditioning disorders.

Glial cells with differential neurite growth-modulating properties probed by atomic force microscopy.

Lateral (L) and medial (M) midbrain astrocytes differ in their ability to support neuritic growth (L, permissive; M, non-permissive) with properties of M glia depending on heparan sulfate (HS). Here we show by atomic force microscopy that the surfaces of formaldehyde-fixed astrocytes differ by conspicuous 250 nm protrusions in L and by a HS-dependent fibrillar network in M glia, thus, demonstrating correlations between cell surface morphology and functional properties.

Quantitative image analysis of temporal changes in tau and neurofilament proteins during the course of acute experimental neurofibrillary degeneration; non-phosphorylated epitopes precede phosphorylation.

Perturbation of the neuronal cytoskeleton represents an integral feature of neurofibrillary tangles which are characteristic neuropathological findings seen in Alzheimer's disease. Microtubule associated protein tau (tau) is considered to be the major component of these lesions although neurofilament proteins also are present. The present study explores the formation of intraneuronal tau and neurofilament protein aggregates using intracisternal administration of aluminum maltolate to rabbits. The time course of the formation of these aggregates and subsequent phosphorylation have been investigated by immunohistochemical methods using a panel of monoclonal antibodies, with quantitation of the staining by image analysis. Neurofilament proteins begin to aggregate by day 1 following aluminum maltolate injection on day 0. Increases in non-phosphorylated neurofilament proteins are observed first, with phosphorylated epitopes being recognized by day 3. Tau follows a similar pattern in that non-phosphorylated epitopes appear to precede phosphorylation. The monoclonal antibody Alz-50 which recognizes a phosphorylation-independent epitope of tau in Alzheimer's disease paired helical filaments, demonstrates positivity in the aluminum maltolate-treated rabbits by day 3. Other tau monoclonal antibodies which recognize phosphorylated tau in paired helical filaments (AT8 and PHF-1) show positive immunostaining on days 6-8. These results indicate that intraneuronal aggregation of cytoskeletal proteins can be initiated by factors other than phosphorylation. However, phosphorylation occurring as a secondary event probably contributes to stabilization of the aggregates.

Laminin inhibits A beta 40 fibril formation promoted by apolipoprotein E4 in vitro.

The aggregation of soluble A beta into insoluble amyloid fibrils is believed to be an important step in the pathogenesis of Alzheimer's disease (AD) and the prevention of this process therefore seems to be a promising strategy for the treatment of AD. Both apolipoprotein E(apoE) and laminin are known to play important roles in the regeneration of the central nervous system and both are known to accumulate in the senile plaques of the AD brains. In the present study, we therefore investigated whether or not laminin has any effect on A beta 40 fibril formation promoted by apoE4 in vitro. A thioflavine-T fluorometric assay and electron microscopic observations using negative staining together demonstrated that laminin inhibits A beta 40 fibril formation in vitro while it also inhibits A beta 40 fibril formation promoted by apoE4. These results suggested that either laminin or its derivatives may thus be effective as therapeutic agents for AD.

Antigenic profile of plaques and neurofibrillary tangles in the amygdala in Down's syndrome: a comparison with Alzheimer's disease.

Most patients with Down's syndrome (DS) undergo a premature cognitive decline with aging, and eventually develop the neuropathologic changes of Alzheimer's disease (AD), including amyloid-containing neuritic plaques, and the formation of neurofibrillary tangles. The amygdala is a focus of marked neuropathologic change in older patients with DS and in AD. We examined the amygdala with immunocytochemical and histochemical methods in 6 cases with DS, ages 19, 20, 27, 29, 56 and 64 years and compared them to 4 cases with AD, ages 54, 76, 77 and 80 years. An antiserum to the A4 amyloid peptide demonstrated amyloid deposition in plaques in all 10 cases. Plaques were also revealed in all cases by the Alcian blue stain for glycosaminoglycans and by the Bielschowsky and Bodian silver stains. An antiserum to alpha-1-antichymotrypsin (ACT) showed plaques in the AD cases and in the 19, 56 and 64 year old DS cases. Neurofibrillary tangles were observed with silver stains only in the older DS and in the AD cases, and not in the 19, 20, 27 and 29 year old DS cases. Likewise, antisera to paired helical filament, to microtubule associated proteins tau and microtubule associated protein-2 (MAP-2), and to ubiquitin, all of which are components of neurofibrillary tangles, reacted with tangles and abnormal neurites only in the older DS and the AD cases. An antiserum to neurofilament epitopes labeled NFTs in the older DS cases and the AD cases, but not in the younger DS cases, except for two intraneuronal NFTs in the 27 year old case.(ABSTRACT TRUNCATED AT 250 WORDS)

Rescue of injury-induced neurofilament loss by BDNF gene transfection in primary septo-hippocampal cell cultures.

We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabeling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated) in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls.

Glutamate AMPA receptors in the fascia dentata of human and kainate rat hippocampal epilepsy.

The present study examined the relationship between the patterns and densities of glutamate AMPA receptor sub-units GluR1 and GluR2/3 in the molecular layer of the fascia dentata and aberrant mossy fiber neoinnervation in human and kainate rat hippocampal epilepsy. Because AMPA sub-units modulate the fast glutamate synaptic transmission, we hypothesized that the AMPA receptor densities would be related to the glutamate-secreting mossy fibers, which could then contribute to seizure generation. In human hippocampal epilepsy, we found that the immunocytochemical labeling of GluR1 and GluR2/3 dendrites was positively related to the densities and spatial locations of the densest, aberrant neo-Timm stained supragranular mossy fibers. We used quantitative densitometry for the mossy fibers. However, the relatively faint and punctate immunocytochemical staining of the receptors did not allow true quantitative densitometry of the dendritic trees because in human epilepsy granule cell densities were decreased on average 50% of normal. Nevertheless, visual observations did confirm spatial relations between dense fascia dentata inner molecular layer mossy fibers and dense AMPA receptor staining. In the outer molecular layer, the mossy fibers were present only in the lower portion, were not densely-stained, and the AMPA receptors were only faintly-labeled. Nevertheless, outer molecular layer AMPA receptor densities were usually present more distally than were the mossy fibers. Experiments were done using intrahippocampal kainate epileptic rats to test the time courses for the changes in mossy fibers and AMPA receptors. The upregulation of inner and outer molecular layer AMPA receptors occurred maximally within 5 days post-kainate injection, prior to any mossy fiber supragranular ingrowth. One hundred and eighty days after ipsilateral kainate the AMPA receptors were increased bilaterally in the inner and outer molecular layers despite the fact that the contralateral aberrant supragranular mossy fibers were minor in comparison to the dense ipsilateral mossy fiber hyperinnervation. These results suggest that in hippocampal epilepsy AMPA receptor numbers increase throughout the length of the molecular layer dendrites; however the AMPA receptor densities are greater in rough relation to the greatest aberrant mossy fiber presynaptic inputs. Interestingly, the receptor upregulation precedes the mossy fiber ingrowth and may play a role in initiating axonal sprouting or in maintaining the aberrant mossy fiber synapses.