[Gene-specific treatment approaches in amyotrophic lateral sclerosis in the present and future]. / Genspezifische Therapieansätze bei amyotropher Lateralsklerose in Gegenwart und Zukunft.

Amyotrophic lateral sclerosis (ALS) is monogenic in up to 10% of cases. Various mutation types result in a loss of function, a gain of toxicity or a combination of both. Due to the continuous development of gene-specific approaches, the treatment of the various ALS forms is no longer a dream. Depending on the underlying mutation type and pathomechanism, different antisense oligonucleotide (ASO)-based or viral strategies are available. The SOD1 and C9ORF72 genes are the most frequently mutated ALS genes in Germany and their mutations most likely predominantly lead to a gain of toxicity. For both genes, specific ASOs were developed binding to the respective mRNAs and leading to their degradation and are now being tested in clinical trials after excellent efficacy in the related ALS mouse models, with promising interim results. For the sporadic form of ALS there are also gene-specific approaches that compensate pathomechanisms and are a promising therapeutic option. In this article, gene-specific therapeutic developments in ALS as well as possible pitfalls and challenges are discussed in detail.

[Genetic architecture of amyotrophic lateral sclerosis and frontotemporal dementia : Overlap and differences]. / Genetische Architektur der amyotrophen Lateralsklerose und frontotemporalen Demenz : Überlappung und Unterschiede.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) overlap not only clinically, but also with respect to shared neuropathology and genes. A large number of novel genes has recently been identified which underlie both diseases, e. g., C9orf72, TARDBP, GRN, TBK1, UBQLN2, VCP, CHCHD10, or SQSTM1. In contrast, other genes are still largely associated with only one of the two diseases, e. g., SOD1 with ALS or MAPT with FTD. These genetic findings indicate a large number of shared mechanisms, yet along with still a certain cell-specific vulnerability. The recently identified genes are not only key to investigate the pathophysiology underlying ALS and FTD, but also the first step in the development of causal gene- or pathway-specific therapies. Mutations in these genes are also found in a substantial share of seemingly "sporadic" ALS and FTD patients. Given the large genetic heterogeneity with more than >25 genes having been identified for ALS and FTD, genetic diagnostics should - after exclusion of C9orf72 repeat expansions - no longer resort to single gene-diagnostics, but rather use next generation sequencing panels or whole exome sequencing.

[Amyotrophic lateral sclerosis. Multisystem degeneration]. / Amyotrophe Lateralsklerose. Eine Multisystemdegeneration.

BACKGROUND: There is increasing evidence that amyotrophic lateral sclerosis (ALS) has to be regarded as multisystem degeneration rather than as purely a motor neuron disease, as it also includes various dnonmotor symptoms. This modern view has been confirmed by neuropathological and imaging findings. OBJECTIVES: To review recent findings supporting the idea of multisystem degeneration and to describe the implications for diagnostics and therapy. METHODS: A discussion of recent clinical, imaging, and neuropathological findings is presented. RESULTS: Symptoms of ALS include not only motor symptoms but also cognitive impairment, oculomotor abnormalities, and extrapyramidal and sensory symptoms. As a neuropathological correlate, a systematic spreading of "transactive response DNA binding protein 43 kDa" (TDP-43) over functionally connected cortical structures has been described. CONCLUSIONS: Nonmotor symptoms are regularly seen in ALS, although they usually do not dominate the clinical picture. Recent neuropathological findings offer new perspectives for diagnostics and therapy in ALS.

[Genetics of amyotrophic lateral sclerosis]. / Genetik der amyotrophen Lateralsklerose.

Amyotrophic lateral sclerosis (ALS) is an aggressive rapidly progressing degeneration of both upper and lower motor neurons. Clinically, ALS is characterized by rapidly progressing atrophy and paresis of the muscles of the extremities. The genetics of ALS have become more complex in the last 5 years. The SOD gene is still very important; however, in recent years mutations in the genes for TDP-43 and FUS were discovered and also a most interesting intronic repeat expansion of the hexanucleotide repeat in C9ORF72 has been shown to be the most common in ALS. There are other quantitatively less relevant genes, which, however, are meaningful for pathogenetic aspects. It is also necessary to know that the phenotypes associated with ALS genetics have expanded.

Truncating mutations in FUS/TLS give rise to a more aggressive ALS-phenotype than missense mutations: a clinico-genetic study in Germany.

BACKGROUND AND PURPOSE: Mutations in the FUS/TLS have been associated with amyotrophic lateral sclerosis (ALS) in a few percent of patients. METHODS: We screened 184 familial (FALS) and 200 sporadic German patients with ALS for FUS/TLS mutations by sequence analysis of exons 5, 6 and 13-15. We compared the phenotypes of patients with different FUS/TLS mutations. RESULTS: We identified three missense mutations p.K510R, p.R514G, p.R521H, and the two truncating mutations p.R495X and p.G478LfsX23 in samples from eight pedigrees. Both truncating mutations were associated with young onset and very aggressive disease courses, whereas the p.R521H, p.R514G and in particular the p.K510R mutation showed a milder phenotype with disease durations ranging from 3 years to more than 26 years, the longest reported for a patient with a FUS/TLS mutation. Also, in a pair of monozygous twins with the p.K510R mutation, a remarkable similar disease course was observed. CONCLUSIONS: Mutations in FUS/TLS account for 8.7% (16 of 184) of FALS in Germany. This is a higher prevalence than reported from other countries. Truncating FUS/TLS mutations result in a more severe phenotype than most missense mutations. The wide phenotypic differences have implications for genetic counselling.

Neuron-specific overexpression of the co-chaperone Bcl-2-associated athanogene-1 in superoxide dismutase 1(G93A)-transgenic mice.

Bcl-2-associated athanogene-1 (BAG1) binds heat-shock protein 70 (Hsp70)/Hsc70, increases intracellular chaperone activity in neurons and proved to be protective in several models for neurodegeneration. Mutations in the superoxide dismutase 1 (SOD1) gene account for approximately 20% of familial amyotrophic lateral sclerosis (ALS) cases. A common property shared by all mutant SOD1 (mtSOD1) species is abnormal protein folding and the propensity to form aggregates. Toxicity and aggregate formation of mutant SOD1 can be overcome by enhanced chaperone function in vitro. Moreover, expression of mtSOD1 decreases BAG1 levels in a motoneuronal cell line. Thus, several lines of evidence suggested a protective role of BAG1 in mtSOD1-mediated motoneuron degeneration. To explore the therapeutic potential of BAG1 in a model for ALS, we generated SOD1G93A/BAG1 double transgenic mice expressing BAG1 in a neuron-specific pattern. Surprisingly, substantially increased BAG1 protein levels in spinal cord neurons did not significantly alter the phenotype of SOD1G93A-transgenic mice. Hence, expression of BAG1 is not sufficient to protect against mtSOD1-induced motor dysfunction in vivo. Our work shows that, in contrast to the in vitro situation, modulation of multiple cellular functions in addition to enhanced expression of a single chaperone is required to protect against SOD1 toxicity, highlighting the necessity of combined treatment strategies for ALS.

Mutant SOD1 detoxification mechanisms in intact single cells.

Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cell-by-cell-analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS.

Cyclin-dependent kinase 5 is an upstream regulator of mitochondrial fission during neuronal apoptosis.

Under physiological conditions, mitochondrial morphology dynamically shifts between a punctuate appearance and tubular networks. However, little is known about upstream signal transduction pathways that regulate mitochondrial morphology. We show that mitochondrial fission is a very early and kinetically invariant event during neuronal cell death, which causally contributes to cytochrome c release and neuronal apoptosis. Using a small molecule CDK5 inhibitor, as well as a dominant-negative CDK5 mutant and RNAi knockdown experiments, we identified CDK5 as an upstream signalling kinase that regulates mitochondrial fission during apoptosis of neurons. Vice versa, our study shows that mitochondrial fission is a modulator contributing to CDK5-mediated neurotoxicity. Thereby, we provide a link that allows integration of CDK5 into established neuronal apoptosis pathways.

The superoxide dismutase1 (SOD1) G93A mutation does not promote neuronal injury after focal brain ischemia and optic nerve transection in mice.

The superoxide dismutase 1 (SOD1)G93A mouse was recently established as transgenic model of amyotrophic lateral sclerosis. We were interested to know whether the SOD1 G93A mutation promotes neuronal injury after intraluminal middle cerebral artery thread occlusion and/or retinal ganglion cell (RGC) axotomy in mice, which are highly reproducible and clinically relevant in vivo models of acute and subacute neuronal degeneration, respectively. In our experiments, G93A mutant SOD1 neither influenced ischemic injury after 90 or 30 min of focal ischemia, nor had an impact on the severity of RGC degeneration after optic nerve transection, when human SOD1 G93A mutant mice were compared to human wild-type SOD1 mice. Our data indicate that the clinically relevant SOD1 G93A mutation, which leads to amyotrophic lateral sclerosis in humans and mice, does not necessarily worsen neuronal degeneration in other pathologies. Thus, the G93A mutation may be counterbalanced in non-motor neurons of young animals, and region-specific and age-related factors may be necessary so that neurodegeneration is re-enforced.

Diffusion tensor imaging for long-term follow-up of corticospinal tract degeneration in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a predominantly clinical and electromyographic diagnosis. Conventional MRI reveals atrophy of the motor system, particularly the pyramidal tract, in the advanced stages but does not provide a sensitive measure of disease progression. Three patients with different principal symptoms of ALS, i.e., with predominant involvement of the upper (UMN) or lower (UMN) motor neurons, or bulbar disease, respectively, underwent serial clinical examination including lung function tests, conventional MRI, and diffusion tensor imaging (DTI). MRI demonstrated changes in of the pyramidal tract without measurable variation on follow-up. The patient with UMN involvement showed remarkable progressive loss of diffusion anisotropy in the pyramidal tract. DTI might be useful, together with clinical follow-up, as an objective morphological marker in therapeutic trials.

Contribution of caspase-8 to apoptosis of axotomized rat retinal ganglion cells in vivo.

We have investigated the role of caspase-8 and its mode of activation during apoptosis of adult rat retinal ganglion cells (RGCs) in vivo. Retinal pro-caspase-8 expression was almost completely restricted to RGCs. Although caspase-8 is known to be involved in death-receptor-dependent apoptosis, measurable caspase-8 activity or even RGC death could be induced by neither tumor necrosis factor-alpha nor Fas ligand injections into unlesioned eyes. However, substantial caspase-8 activation could be detected after optic nerve transection as shown by a fluorogenic activity assay and Western blot analysis. Intravitreal injection of caspase-8 inhibitors significantly attenuated degeneration of RGCs and reduced the number of RGCs showing caspase-3 activation. A late peak of caspase-8 activity and additive protective effects of caspase-8 and -9 inhibition on axotomized RGCs place caspase-8 in our model rather late in the apoptosis cascade, possibly after the onset of mitochondrial dysfunction.

Cyclin-dependent kinase 5 (CDK5) and neuronal cell death.

Many neurological disorders like Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis (ALS) or stroke have in common a definite loss of CNS neurons due to apoptotic or necrotic neuronal cell death. Previous studies suggested that proapoptotic stimuli may trigger an abortive and, therefore, eventually fatal cell cycle reentry in postmitotic neurons. Neuroprotective effects of small molecule inhibitors of cyclin-dependent kinases (CDKs), which are key regulators of cell cycle progression, support the cell cycle theory of neuronal apoptosis. However, growing evidence suggests that deregulated CDK5, which is not involved in cell cycle control, rather than cell cycle relevant members of the CDK family, promotes neuronal cell death. Here we summarize the current knowledge about the involvement of CDK5 in neuronal cell death and discuss possible up- or downstream partners of CDK5. Moreover, we discuss potential therapeutic options that might arise from the identification of CDK5 as an important upstream element of neuronal cell death cascades.

Kir channels in the CNS: emerging new roles and implications for neurological diseases.

Inwardly rectifying potassium (Kir) channels have long been regarded as transmembrane proteins that regulate the membrane potential of neurons and that are responsible for [K(+)] siphoning in glial cells. The subunit diversity within the Kir channel family is growing rapidly and this is reflected in the multitude of roles that Kir channels play in the central nervous system (CNS). Kir channels are known to control cell differentiation, modify CNS hormone secretion, modulate neurotransmitter release in the nigrostriatal system, may act as hypoxia-sensors and regulate cerebral artery dilatation. The increasing availability of genetic mouse models that express inactive Kir channel subunits has opened new insights into their role in developing and adult mammalian tissues and during the course of CNS disorders. New aspects with respect to the role of Kir channels during CNS cell differentiation and neurogenesis are also emerging. Dysfunction of Kir channels in animal models can lead to severe phenotypes ranging from early postnatal death to an increased susceptibility to develop epileptic seizures. In this review, we summarize the in vivo data that demonstrate the role of Kir channels in regulating morphogenetic events, such as the proliferation, differentiation and survival of neurons and glial cells. We describe the way in which the gating of Kir channel subunits plays an important role in polygenic CNS diseases, such as white matter disease, epilepsy and Parkinson's disease.

Transection of the optic nerve in rats: studying neuronal death and survival in vivo.

Transection of the optic nerve (ON) in the adult rat, as a model of fiber tract lesion in the adult mammalian CNS, results in delayed, mainly apoptotic death of 80--90% of retinal ganglion cells (RGCs) within 14 days post-lesion. Because of good surgical accessibility of the retina and the optic nerve, the retino-tectal projection represents not only a convenient model to study the molecular mechanisms underlying neuronal death but also serves as a suitable system for investigating potential neuroprotective agents in vivo. In the present report, we provide a detailed protocol for this model including retrograde labeling of RGCs, ON lesion, assessment of the number of surviving neurons, and tissue preparation for several standard techniques like immunohistochemistry, reverse transcription--polymerase chain reaction (RT--PCR), enzyme assays and Immunoblot.

Reduction of potassium currents and phosphatidylinositol 3-kinase-dependent AKT phosphorylation by tumor necrosis factor-(alpha) rescues axotomized retinal ganglion cells from retrograde cell death in vivo.

Tumor-necrosis-factor-alpha (TNF-alpha) prevented secondary death of retinal ganglion cells (RGCs) after axotomy of the optic nerve in vivo. This RGC rescue was confirmed in vitro in a mixed retinal culture model. In accordance with our previous findings, TNF-alpha decreased outward potassium currents in RGCs. Antagonism of the TNF-alpha-induced decrease in outward potassium currents with the potassium channel opener minoxidilsulfate (as verified by electrophysiology) abolished neuroprotection. Western blot analysis revealed an upregulation of phospho-Akt as a consequence of TNF-alpha-induced potassium current reduction. Inhibition of the phosphatidylinositol 3-kinase-Akt pathway with wortmannin decreased TNF-alpha-promoted RGC survival. These data point to a functionally relevant cytokine-dependent neuroprotective signaling cascade in adult CNS neurons.

Degeneration of axotomized retinal ganglion cells as a model for neuronal apoptosis in the central nervous system - molecular death and survival pathways.

Programmed cell death (PCD) or apoptosis is a phenomenon important for proper development and morphological as well as functional fine tuning of the nervous system. In the past two decades it became evident that the same apoptotic machinery, which has crucial functions in during development, can be reactivated under pathological circumstances in the adult nervous system and contribute to neuronal cell loss due to various neurological disorders like ischemic stroke, neurodegenerative diseases or brain traumata. In this review, we present the optic nerve transection paradigm as a valuable model for investigation of apoptotic neuronal cell death in the central nervous system (CNS). We review and summarize the most important discoveries regarding molecular pathways and mechanisms of neuronal apoptosis during the past few years, and outline contributions that have been made investigating the death of retinal ganglion cells (RGCs) following transection of the optic nerve.

Brain-derived neurotrophic factor-mediated neuroprotection of adult rat retinal ganglion cells in vivo does not exclusively depend on phosphatidyl-inositol-3'-kinase/protein kinase B signaling.

The neurotrophin brain-derived neurotrophic factor (BDNF) serves as a survival, mitogenic, and differentiation factor in both the developing and adult CNS and PNS. In an attempt to identify the molecular mechanisms underlying BDNF neuroprotection, we studied activation of two potentially neuroprotective signal transduction pathways by BDNF in a CNS trauma model. Transection of the optic nerve (ON) in the adult rat induces secondary death of retinal ganglion cells (RGCs). Repeated intraocular injections of BDNF prevent the degeneration of RGCs 14 d after ON lesion most likely by inhibition of apoptosis. Here, we report that BDNF activates both protein kinase B (PKB) via a phosphatidyl-inositol-3'-kinase (PI-3-K)-dependent mechanism and the mitogen-activated protein kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2. Furthermore, we provide evidence that BDNF suppresses cleavage and enzymatic activity of the neuronal cell death effector caspase-3. Distinct from our recent study in which inhibition of the PI-3-K/PKB pathway attenuated the survival-promoting action of insulin-like growth factor-I on axotomized RGCs (Kermer et al., 2000), it does not in the case of BDNF. Thus, we assume that BDNF does not depend on a single signal transduction pathway exerting its neuroprotective effects on lesioned CNS neurons.

NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex.

NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.

Sublethal oxygen-glucose deprivation alters hippocampal neuronal AMPA receptor expression and vulnerability to kainate-induced death.

Recent studies have suggested that rats subjected to transient global brain ischemia develop depressed expression of GluR-B in CA1 hippocampal neurons. The present study was performed to determine whether a similar change in AMPA receptor expression could be triggered in vitro by sublethal oxygen-glucose deprivation in rat hippocampal neuronal cultures. mRNA was extracted from individual hippocampal neurons via patch electrodes and amplified by RT-PCR 24-48 hr after sublethal oxygen-glucose deprivation. Compared with controls, insulted neurons expressed increased levels of GluR-D flop. As an indication that this change in receptor expression was functionally significant, insulted cultures exhibited increased AMPA- or kainate-induced 45Ca2+ accumulation sensitive to Joro spider toxin and increased vulnerability to kainate-induced death. These data support the hypothesis that exposure to ischemia may enhance subsequent hippocampal neuronal vulnerability to AMPA receptor-mediated excitotoxicity by modifying the relative expression of AMPA receptor subunits in a manner that promotes Ca2+ permeability.