In superior cervical ganglia neurons, frequency and velocity of neurofilament movements were reduced both in anterograde and retrograde direction [ 30 ]. In a mouse model with the human NS mutation in the KIF5A gene cortical neurons revealed an increased retrograde velocity of neurofilament M transport, whereas anterograde velocity was not affected. The frequency of anterograde and retrograde movements was decreased [ 31 ]. But probably neurofilament is not the only important cargo transported by KIF5A.
In this work, we established primary cell cultures of motor and sensory neurons of constitutive KIF5A KO mice and characterized vitality, morphology, and function. By live cell imaging, we revealed an axonal transport defect of mitochondria. These results can improve insights into mechanisms underlying the length-dependent axonal degeneration and selective damage of motor neurons in SPG For histological analysis embryos were dissected from pregnant mice at embryonic day E After dehydration tissues were embedded in paraffin.
For morphological assessment of spinal motor neurons the size of cell bodies and nuclei were analyzed in at least 36 cells from three independent experiments. Embryos were isolated from pregnant mice at E The trypsin reaction 0. Tissues were triturated.
Fifty percent of the medium was replaced every day. Genomic DNA from mouse tissues was isolated by standard methods. Then, every day the number of surviving cells in ten fields of view 1. The percentage of surviving cells was calculated for every day in culture. The results from at least four independent experiments were pooled. So for each genotype at least 11 individuals were counted, and are given as mean and standard error of mean.
After treatment with 0. Nuclei were stained with Hoechst ,, Sigma-Aldrich. All images were obtained using identical camera, microscope, and imaging criteria such as gain, brightness and contrast, and exposure time. Morphological assessment of axonal swellings was performed in three independent experiments, for each genotype at least cells were analyzed. The results are given as mean and standard deviation. For outgrowth analysis, axons of motor neurons were identified via positive anti-phospho-tau staining, dendrites via positive anti-MAP2ab staining.
The following outgrowth parameters were measured with the Neurolucida 8 software MBF Bioscience, Williston, USA , and are given as mean and standard deviation: longest axonal branch, total axon length including all branches , longest dendrite, total dendrite length of one cell , number of axonal branches, number of dendrites, and cell body area.
An example for the outgrowth analysis is shown in Online Resource 1. In sensory neurons, anti-phospho-tau positive neurite length total neurite length, longest neurite was measured. Results from three independent experiments were pooled, in summary at least cells of each genotype were analyzed for statistical differences by ANOVA followed by Bonferroni correction SPSS Altogether, at least 53 mitochondria in 18 different cells were counted for each genotype.
Second, the length of mitochondria from seven independent experiments at least mitochondria for each genotype was measured with Neurolucida 8 software MBF Bioscience. The results are indicated as mean and standard deviation SD. Mitochondria were stained and visualized as described above.
For statistical analysis of mitochondrial movements ten independent experiments were performed. Mice lacking KIF5A have a severe phenotype with death shortly after birth [ 29 ]. Additionally, muscle tissue appeared normal in all three genotypes exemplary images are shown in Online Resource 2. Motor and sensory neuron cell cultures were obtained from the lumbar spinal cord and the adjacent DRGs, dissected from day E Cells were identified as motor and sensory neurons by morphology and positive anti-islet, anti-AChT, anti-MAP2ab, and anti-phospho-tau staining.
Typical examples are shown in Online Resource 3. In sensory neurons, there was no significant difference between the three genotypes Fig. Motor neurons are indicated in continuous lines, sensory neurons in broken lines. In sensory neurons, there was no significant difference between the three genotypes at any time point. In total, at least 11 mice were analyzed per genotype. Mean and SEM are given.
In sensory neurons, no gross morphological abnormalities were observed data not shown. Motor neurons were stained with an antibody against neurofilament L white. Since in HSP, the motor neurons with the longest axons are preferentially affected, we assessed the influence of KIF5A knockout on process outgrowth. Dendrites were stained by an anti-MAP2ab antibody. Similar results were obtained for the longest axonal branch data not shown. Results are given as mean; due to clarity standard deviation indicators have been omitted.
Measured values were linearly interpolated. For each genotype more than cells from three independent experiments were analyzed. Results are given as mean and standard deviation. The number of neurites was not altered between the genotypes Fig. As axonal transport disturbances of mitochondria may lead to a reduced number of organelles in a length-dependent manner, we counted mitochondria in proximal and distal parts of the axon.
Since damaged mitochondria often are fragmented and present with a more round appearance whereas viable mitochondria show an elongated shape [ 32 , 33 ] we assessed mitochondrial morphology as a potential indicator of impaired mitochondrial function. To assess mitochondrial transport as a critical factor of energy supply especially in distal parts of the axon and at the synapse, we analyzed mitochondrial movements in axons of motor neurons both in anterograde and retrograde direction using time-lapse imaging.
Axonal transport of mitochondria in motor neurons. Mean and standard deviation are given. Results from ten independent experiments were summarized. In summary, we observed impaired axonal transport, reduced axonal outgrowth, and reduced survival of primary motor neurons lacking KIF5A. However, several observations argue for axonopathy as the primary problem caused by the lack of KIF5A.
Axon and dendrite growth is not generally impaired but becomes obvious only when processes exceed a critical length. Furthermore, the observation of an increased number of axonal swellings in cultured motor neurons lacking KIF5A points to primary axonal impairment and is in line with autopsy findings of spastic paraplegia, e. For SPG10, no pathoanatomical studies are available.
Defects of anterograde and retrograde transport as observed in our study but also in squid and drosophila models lacking kinesin-1 are in line with primary axonopathy. To assess axonal transport as a potentially critical factor in the extreme anatomy of motor neurons we tracked mitochondrial movements in motor neurons with time-lapse microscopy.
Interestingly, we found a significant reduction of transport velocity in both directions, anterograde and retrograde. Perturbance of anterograde transport seems to be plausible for a motor protein involved in fast anterograde transport, whereas disturbance of retrograde transport is surprising at first glance. In accordance with our findings, involvement of kinesin-1 both in anterograde and retrograde transport has been shown in three additional models: Perfusion of squid axoplasms with an antibody against KHC inhibited rate and number of anterogradely and retrogradely moving organelles [ 35 , 36 ].
Drosophila flies lacking the KIF5A homologue KHC also have an axonal transport deficit for mitochondria both in the anterograde and retrograde direction [ 37 , 38 ]. The human NS mutation of the KIF5A gene in a mouse model causes a reduced frequency of neurofilament transport in both directions, whereas transport velocity is increased in retrograde and normal in anterograde direction [ 31 ].
Different scenarios could explain the axonal transport impairment in both directions. First, if mitochondria are produced in or near the cell body, they have to be transported properly to the periphery before they can head back to the cell body. This fact alone seems not sufficient to explain our results since the number of mitochondria in the periphery is not altered.
Second, the retrograde motor protein dynein is synthesized in the cell body and needs to be transported to distal parts of the axon before participating in retrograde transport. This hypothesis is supported by the dependence of anterograde dynein transport on kinesin-1 [ 36 , 39 ].
Third, KIF5A could act as a biochemical or biophysical activator of dynein in axons. In the recent years, the concept of cargoes concomitantly bound to motors for both directions has been accepted [ 40 ]. The exact mode of motor coordination and regulation of axonal transport direction and velocity is still unresolved. Experimental data examining whether the number of active motors influences transport velocity and length are controversial [ 41 — 43 ].
The number of other factors implicated in kinesin activity and transport dynamics is increasing steadily and depends on the cargo [ 45 ]. The interdependence of KIF5A and dynein is also enforced by the fact that disruption of dynein function by using RNA interference inhibited neurofilament transport both in anterograde and retrograde direction [ 30 ].
Mitochondrial transport seems to be influenced amongst other factors by mitochondrial membrane potential [ 46 ], nerve growth factor [ 47 ], dynamin-related protein-1 [ 48 ], and phosphatidylinositol [ 49 ]. But a global plan of the interactions has not yet emerged. Beyond mitochondria for power supply and neurofilaments as part of the cytoskeleton, the members of the kinesinfamily transport a variety of other cargoes, also including tubulin [ 24 ], synaptic vesicles [ 22 , 23 ], and lysosomes [ 21 ].
These cargoes could also be critical for neuronal survival, outgrowth, and synaptic function. However, a detailed understanding of the full repertoire of cargoes transported by KIF5A is not yet available and requires further work. In addition, axon length has to exceed a critical length until the outgrowth defect gets obvious.
This nicely parallels the situation in patients with SPG10 where the longest axons of the corticospinal tract innervating the legs are mostly affected, whereas the shorter axons to the arms are mainly spared. If the length of processes is critical, the involvement of other cell types with long processes has to be expected as well. Indeed, in sensory neurons which have axons of similar length as motor neurons we found reduced outgrowth as well, even though to a lesser extent than in motor neurons.
This would also be concordant to the human disease: patients with SPG10 may have additional sensory deficits, but much less severe than motor affection. The cause of primary involvement of motor neurons is not known but may be due to special functions executed by specific linking or binding proteins, higher need of common cell functions, e.
However, a high variability in the age at onset and disease severity even within one family has been reported for the HSPs, including SPG10, so additional factors seem to influence the development of the disease. Whether KIF5A mutations cause SPG10 by a lack of functional kinesin haploinsuffiency or by a dominant negative effect is not quite clear. The majority of mutations in SPG10 patients are missense mutations in the motor domain of kinesin which is responsible for binding of kinesin to microtubules and its movement along microtubules.
Indeed, functional in silico analyses found that kinesin with different missense mutations causing SPG10 in humans leads to a reduced binding to microtubules or reduced transport velocity, respectively [ 28 ]. In vivo, the kinesin-1 complex is a heterotetramer made up of two kinesin heavy chains and two light chains.
Whether this drastic decrease of functional kinesin is causing disease or, alternatively, microtubules become blocked from dysfunctional kinesin causing a traffic jam or, as a third scenario, abnormal kinesin binds to cargo and blocks its processing, remains to be established. Our animal model hints at an involvement of mitochondrial transport in the pathogenesis of some forms of HSPs. This might be a link to other HSP forms that are caused by mitochondrial dysfunction, e.
In summary, our results point out a role of KIF5A in process outgrowth and axonal transport of mitochondria, affecting motor neurons more severely than sensory neurons. Spastische Spinalparalysen. For example, genomic copy number changes may initially result in an altered protein expression, but this effect might be replaced by other mechanisms during later phases of tumour development. In conclusion, we demonstrated a novel approach providing candidate genes of potential clinical and biological relevance.
Immunohistochemical analyses revealed several candidates correlating with clinico-pathological factors that are markers for prostate cancer-specific survival. The impact of the candidates on prostate carcinogenesis and their potential clinical application remain to be analysed in further detailed studies.
We are indebted to Carsten Sticht for statistical support, David Trick for reviewing of tissue microarray sections, Corinna Weidt and Felix Engel for critical reading of the manuscript. Read article at publisher's site DOI : PLoS One , 15 8 :e, 28 Aug Oncogene , 39 11 , 20 Jan Kulik G. Cancers Basel , 11 3 , 13 Mar Front Oncol , , 18 Sep Acta Pharmacol Sin , 39 11 , 19 Jun Review Free to read.
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Revised Sep 20; Accepted Oct Copyright , Cancer Research UK. This article has been cited by other articles in PMC. Go to:. Patient material Formalin-fixed, paraffin-embedded primary prostate adenocarcinomas resected between and were diagnosed in the Division of cellular and molecular pathology at the German Cancer Research Centre, Heidelberg. Table 1 Clinico-pathological features of patients a, b. Clinico-pathological factor Classification Tumour no. Open in a separate window.
Both tumour material and BPH material could be analysed from 82 patients. Additionally, carcinoma was analysable in 49 cases and BPH in another 44 cases. Analysis of genomic and expression data sets To reveal the most abundant chromosomal imbalances detected in prostate carcinoma, we performed an exhaustive search of six CGH studies, including a total of primary prostate cancers Sattler et al , ; Alers et al , ; Verdorfer et al , ; Zitzelsberger et al , ; Steiner et al , ; Wolter et al , ; Paris et al , as well as one array CGH study of 16 primary prostate cancers Paris et al , Construction of a prostate carcinoma tissue microarray Tissue microarrays were constructed as originally described Kononen et al , Statistical analyses Statistical analyses were performed using the statistical software package SPSS Selection of candidate genes by correlation of genomic and expression data sets To establish a list of candidate genes relevant for prostate carcinogenesis, we first performed an exhaustive search of six previously published CGH analyses primary prostate cancers and one array CGH analysis 16 primary prostate cancers see Material and Methods.
Subcellular expression pattern of candidate proteins Examples of typical staining patterns from the antibodies used are shown in Figure 1. Figure 1. Correlation of protein expression and clinico-pathological parameters using a prostate cancer tissue microarray Univariate analyses In order to test whether the expression of the selected proteins correlates with clinico-pathological parameters, we constructed a tissue microarray.
Table 3 Immunostaining of all analysed proteins in BPH and prostate tumour samples. Figure 2. Table 4 Correlation between clinico-pathological factors and positive immunostaining of all proteins analysed. Not all BPH samples correspond to the same patients as the tumour samples. Multivariate analyses Logistic regression was used to predict the outcome variables Gleason score Gs2—6 vs Gs7—9 and pT-stage organ-confined vs extraprostatic Table 5. Supplementary Table 2. Molecular cytogenetic analysis of prostatic adenocarcinomas from screening studies: early cancers may contain aggressive genetic features.
Am J Pathol. Fatty acid synthase: a metabolic oncogene in prostate cancer? J Cell Biochem. Constitutively active protein phosphatase 1alpha causes Rb-dependent G1 arrest in human cancer cells. Curr Biol. High-throughput tissue microarray analysis of c-myc activation in chronic liver diseases and hepatocellular carcinoma.
Hum Pathol. Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas—a possible mechanism for altering the nmH1 activity. Prostate cancer diagnosis and management. High levels of nm23 expression are related to cell proliferation in human prostate cancer. Cancer Res. Biochem J. Cancer statistics, CA Cancer J Clin. Detection of c-myc oncogene amplification and chromosomal anomalies in metastatic prostatic carcinoma by fluorescence in situ hybridization.
Androgen receptor activation by G s signaling in prostate cancer cells. J Biol Chem. Expression of nmH1 and nmH2 proteins in prostate carcinoma. Jpn J Cancer Res. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat Med. Serine-threonine protein phosphatase inhibitors: development of potential therapeutic strategies. J Med Chem.
Tissue microarrays TMAs for high-throughput molecular pathology research. Int J Cancer. High-resolution analysis of paraffin-embedded and formalin-fixed prostate tumors using comparative genomic hybridization to genomic microarrays. Seven-transmembrane receptors.
Nat Rev Mol Cell Biol. Loss of p53 and c-myc overrepresentation in stage T N M 0 prostate cancer are potential markers for cancer progression. Mod Pathol. Meta-analysis of microarrays: interstudy validation of gene expression profiles reveals pathway dysregulation in prostate cancer. Fatty acid synthase expression defines distinct molecular signatures in prostate cancer. Mol Cancer Res. Interferon-gamma and its functional receptors overexpression in benign prostatic hyperplasia and prostatic carcinoma: parallelism with c-myc and p53 expression.
Eur Cytokine Netw. Clinical significance of alterations of chromosome 8 in high-grade, advanced, nonmetastatic prostate carcinoma. J Natl Cancer Inst. Comparative genomic hybridization reveals DNA copy number gains to frequently occur in human prostate cancer. Immunohistochemical detection of a fatty acid synthase OA as a predictor of progression of prostate cancer.
Metastasis suppressors alter the signal transduction of cancer cells. Nat Rev Cancer. Gain in chromosome 8q correlates with early progression in hormonal treated prostate cancer. Eur Urol. Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer. Tissue microarrays for rapid linking of molecular changes to clinical endpoints.
Clinical significance of alterations of chromosome 8 detected by fluorescence in situ hybridization analysis in pathologic organ-confined prostate cancer. Genes Chromosomes Cancer. Combined study of prostatic carcinoma by classical cytogenetic analysis and comparative genomic hybridization.
Int J Oncol. Chromosomal changes in incidental prostatic carcinomas detected by comparative genomic hybridization. Chromosomal changes during development and progression of prostate adenocarcinomas. Smart citations by scite. The number of the statements may be higher than the number of citations provided by EuropePMC if one paper cites another multiple times or lower if scite has not yet processed some of the citing articles.
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