Development of a new hazard scoring system in primary neuronal cell cultures for drug-induced acute neuronal toxicity identification in early drug discovery.

We investigated drug-induced acute neuronal electrophysiological changes using Micro-Electrode arrays (MEA) to rat primary neuronal cell cultures. Data based on 6-key MEA parameters were analyzed for plate-to-plate vehicle variability, effects of positive and negative controls, as well as data from over 100 reference drugs, mostly known to have pharmacological phenotypic and clinical outcomes. A Least Absolute Shrinkage and Selection Operator (LASSO) regression, coupled with expert evaluation helped to identify the 6-key parameters from many other MEA parameters to evaluate the drug-induced acute neuronal changes. Calculating the statistical tolerance intervals for negative-positive control effects on those 4-key parameters helped us to develop a new weighted hazard scoring system on drug-induced potential central nervous system (CNS) adverse effects (AEs). The weighted total score, integrating the effects of a drug candidate on the identified six-pivotal parameters, simply determines if the testing compound/concentration induces potential CNS AEs. Hereto, it uses four different categories of hazard scores: non-neuroactive, neuroactive, hazard, or high hazard categories. This new scoring system was successfully applied to differentiate the new compounds with or without CNS AEs, and the results were correlated with the outcome of in vivo studies in mice for one internal program. Furthermore, the Random Forest classification method was used to obtain the probability that the effect of a compound is either inhibitory or excitatory. In conclusion, this new neuronal scoring system on the cell assay is actively applied in the early de-risking of drug development and reduces the use of animals and associated costs.

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer's disease.

BACKGROUND: Microglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer's disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs). METHODS: In this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo. RESULTS: We proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-ß plaques in vivo in a model of AD. CONCLUSIONS: This study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

From Junk to Function: LncRNAs in CNS Health and Disease.

Recent advances in RNA sequencing technologies helped to uncover the existence of tens of thousands of long non-coding RNAs (lncRNAs) that arise from the dark matter of the genome. These lncRNAs were originally thought to be transcriptional noise but an increasing number of studies demonstrate that these transcripts can modulate protein-coding gene expression by a wide variety of transcriptional and post-transcriptional mechanisms. The spatiotemporal regulation of lncRNA expression is particularly evident in the central nervous system, suggesting that they may directly contribute to specific brain processes, including neurogenesis and cellular homeostasis. Not surprisingly, lncRNAs are therefore gaining attention as putative novel therapeutic targets for disorders of the brain. In this review, we summarize the recent insights into the functions of lncRNAs in the brain, their role in neuronal maintenance, and their potential contribution to disease. We conclude this review by postulating how these RNA molecules can be targeted for the treatment of yet incurable neurological disorders.

Missing lnc(RNAs) in Alzheimer's Disease?

With the ongoing demographic shift towards increasingly elderly populations, it is estimated that approximately 150 million people will live with Alzheimer's disease (AD) by 2050. By then, AD will be one of the most burdensome diseases of this and potentially next centuries. Although its exact etiology remains elusive, both environmental and genetic factors play crucial roles in the mechanisms underlying AD neuropathology. Genome-wide association studies (GWAS) identified genetic variants associated with AD susceptibility in more than 40 different genomic loci. Most of these disease-associated variants reside in non-coding regions of the genome. In recent years, it has become clear that functionally active transcripts arise from these non-coding loci. One type of non-coding transcript, referred to as long non-coding RNAs (lncRNAs), gained significant attention due to their multiple roles in neurodevelopment, brain homeostasis, aging, and their dysregulation or dysfunction in neurological diseases including in AD. Here, we will summarize the current knowledge regarding genetic variations, expression profiles, as well as potential functions, diagnostic or therapeutic roles of lncRNAs in AD. We postulate that lncRNAs may represent the missing link in AD pathology and that unraveling their role may open avenues to better AD treatments.

Age-dependent SMN expression in disease-relevant tissue and implications for SMA treatment.

BACKGROUNDSpinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein. New SMN-enhancing therapeutics are associated with variable clinical benefits. Limited knowledge of baseline and drug-induced SMN levels in disease-relevant tissues hinders efforts to optimize these treatments.METHODSSMN mRNA and protein levels were quantified in human tissues isolated during expedited autopsies.RESULTSSMN protein expression varied broadly among prenatal control spinal cord samples, but was restricted at relatively low levels in controls and SMA patients after 3 months of life. A 2.3-fold perinatal decrease in median SMN protein levels was not paralleled by comparable changes in SMN mRNA. In tissues isolated from nusinersen-treated SMA patients, antisense oligonucleotide (ASO) concentration and full-length (exon 7 including) SMN2 (SMN2-FL) mRNA level increases were highest in lumbar and thoracic spinal cord. An increased number of cells showed SMN immunolabeling in spinal cord of treated patients, but was not associated with an increase in whole-tissue SMN protein levels.CONCLUSIONSA normally occurring perinatal decrease in whole-tissue SMN protein levels supports efforts to initiate SMN-inducing therapies as soon after birth as possible. Limited ASO distribution to rostral spinal and brain regions in some patients likely limits clinical response of motor units in these regions for those patients. These results have important implications for optimizing treatment of SMA patients and warrant further investigations to enhance bioavailability of intrathecally administered ASOs.FUNDINGSMA Foundation, SMART, NIH (R01-NS096770, R01-NS062869), Ionis Pharmaceuticals, and PTC Therapeutics. Biogen provided support for absolute real-time RT-PCR.

Motor neuron loss in SMA is not associated with somal stress-activated JNK/c-Jun signaling.

A pathological hallmark of spinal muscular atrophy (SMA) is severe motor neuron (MN) loss, which results in muscle weakness and often infantile or childhood mortality. Although it is well established that deficient expression of survival motor neuron (SMN) protein causes SMA, the molecular pathways that execute MN cell death are poorly defined. The c-Jun NH2-terminal kinases (JNKs) are stress-activated kinases with multiple substrates including c-Jun, which can be activated during neuronal injury and neurodegenerative disease leading to neuronal apoptosis. Recently, increased JNK-c-Jun signaling was reported in SMA raising the possibility that JNK inhibitors could be a novel treatment for this disease. We examined JNK-c-Jun activity in SMA mouse and human cultured cells and tissues. Anisomycin treatment of human SMA fibroblasts and sciatic nerve ligation in SMA mice provoked robust phosphorylated-c-Jun (p-c-Jun) expression indicating that SMN-deficiency does not prevent activation of the stress-induced JNK-c-Jun signaling pathway. Despite retained capacity to activate JNK-c-Jun, we observed no basal increase of p-c-Jun levels in SMA compared to control cultured cells, human or mouse spinal cord tissues, or mouse MNs during the period of MN loss in severe SMA model mice. In both controls and SMA, ~50% of α-MN nuclei express p-c-Jun with decreasing expression during the early postnatal period. Together these studies reveal no evidence of stress-activated JNK-c-Jun signaling in MNs of SMA mice or human tissues, but do highlight the important role of JNK-c-Jun activity during normal MN development raising caution about JNK antagonism in this pediatric neuromuscular disease.

Genetically Engineered iPSC-Derived FTDP-17 MAPT Neurons Display Mutation-Specific Neurodegenerative and Neurodevelopmental Phenotypes.

Tauopathies such as frontotemporal dementia (FTD) remain incurable to date, partially due to the lack of translational in vitro disease models. The MAPT gene, encoding the microtubule-associated protein tau, has been shown to play an important role in FTD pathogenesis. Therefore, we used zinc finger nucleases to introduce two MAPT mutations into healthy donor induced pluripotent stem cells (iPSCs). The IVS10+16 mutation increases the expression of 4R tau, while the P301S mutation is pro-aggregant. Whole-transcriptome analysis of MAPT IVS10+16 neurons reveals neuronal subtype differences, reduced neural progenitor proliferation potential, and aberrant WNT/SHH signaling. Notably, these neurodevelopmental phenotypes could be recapitulated in neurons from patients carrying the MAPT IVS10+16 mutation. Moreover, the additional pro-aggregant P301S mutation revealed additional phenotypes, such as an increased calcium burst frequency, reduced lysosomal acidity, tau oligomerization, and neurodegeneration. This series of iPSCs could serve as a platform to unravel a potential link between pathogenic 4R tau and FTD.

HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease.

Peripheral nerve axons require a well-organized axonal microtubule network for efficient transport to ensure the constant crosstalk between soma and synapse. Mutations in more than 80 different genes cause Charcot-Marie-Tooth disease, which is the most common inherited disorder affecting peripheral nerves. This genetic heterogeneity has hampered the development of therapeutics for Charcot-Marie-Tooth disease. The aim of this study was to explore whether histone deacetylase 6 (HDAC6) can serve as a therapeutic target focusing on the mutant glycyl-tRNA synthetase (GlyRS/GARS)-induced peripheral neuropathy. Peripheral nerves and dorsal root ganglia from the C201R mutant Gars mouse model showed reduced acetylated α-tubulin levels. In primary dorsal root ganglion neurons, mutant GlyRS affected neurite length and disrupted normal mitochondrial transport. We demonstrated that GlyRS co-immunoprecipitated with HDAC6 and that this interaction was blocked by tubastatin A, a selective inhibitor of the deacetylating function of HDAC6. Moreover, HDAC6 inhibition restored mitochondrial axonal transport in mutant GlyRS-expressing neurons. Systemic delivery of a specific HDAC6 inhibitor increased α-tubulin acetylation in peripheral nerves and partially restored nerve conduction and motor behaviour in mutant Gars mice. Our study demonstrates that α-tubulin deacetylation and disrupted axonal transport may represent a common pathogenic mechanism underlying Charcot-Marie-Tooth disease and it broadens the therapeutic potential of selective HDAC6 inhibition to other genetic forms of axonal Charcot-Marie-Tooth disease.

The Antisense Transcript SMN-AS1 Regulates SMN Expression and Is a Novel Therapeutic Target for Spinal Muscular Atrophy.

The neuromuscular disorder spinal muscular atrophy (SMA), the most common inherited killer of infants, is caused by insufficient expression of survival motor neuron (SMN) protein. SMA therapeutics development efforts have focused on identifying strategies to increase SMN expression. We identified a long non-coding RNA (lncRNA) that arises from the antisense strand of SMN, SMN-AS1, which is enriched in neurons and transcriptionally represses SMN expression by recruiting the epigenetic Polycomb repressive complex-2. Targeted degradation of SMN-AS1 with antisense oligonucleotides (ASOs) increases SMN expression in patient-derived cells, cultured neurons, and the mouse central nervous system. SMN-AS1 ASOs delivered together with SMN2 splice-switching oligonucleotides additively increase SMN expression and improve survival of severe SMA mice. This study is the first proof of concept that targeting a lncRNA to transcriptionally activate SMN2 can be combined with SMN2 splicing modification to ameliorate SMA and demonstrates the promise of combinatorial ASOs for the treatment of neurogenetic disorders.

Spinal Muscular Atrophy Therapeutics: Where do we Stand?

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder pathologically characterized by the degeneration of motor neurons in the spinal cord and muscle atrophy. Motor neuron loss often results in severe muscle weakness causing affected infants to die before reaching 2 years of age. Patients with milder forms of SMA exhibit slowly progressive muscle weakness over many years. SMA is caused by the loss of SMN1 and the retention of at least 1 copy of a highly homologous SMN2. An alternative splicing event in the pre-mRNA arising from SMN2 results in the production of low levels of functional SMN protein. To date, there are no effective treatments available to treat patients with SMA. However, over the last 2 decades, the development of SMA mouse models and the identification of therapeutic targets have resulted in a promising drug pipeline for SMA. Here, we highlight some of the therapeutic strategies that have been developed to activate SMN2 expression, modulate splicing of the SMN2 pre-mRNA, or replace SMN1 by gene therapy. After 2 decades of translational research, we now stand within reach of a treatment for SMA.

A dominant mutation in FBXO38 causes distal spinal muscular atrophy with calf predominance.

Spinal muscular atrophies (SMAs) are a heterogeneous group of inherited disorders characterized by degeneration of anterior horn cells and progressive muscle weakness. In two unrelated families affected by a distinct form of autosomal-dominant distal SMA initially manifesting with calf weakness, we identified by genetic linkage analysis and exome sequencing a heterozygous missense mutation, c.616T>C (p.Cys206Arg), in F-box protein 38 (FBXO38). FBXO38 is a known coactivator of the transcription factor Krüppel-like factor 7 (KLF7), which regulates genes required for neuronal axon outgrowth and repair. The p.Cys206Arg substitution did not alter the subcellular localization of FBXO38 but did impair KLF7-mediated transactivation of a KLF7-responsive promoter construct and endogenous KLF7 target genes in both heterologously expressing human embryonic kidney 293T cells and fibroblasts derived from individuals with the FBXO38 missense mutation. This transcriptional dysregulation was associated with an impairment of neurite outgrowth in primary motor neurons. Together, these results suggest that a transcriptional regulatory pathway that has a well-established role in axonal development could also be critical for neuronal maintenance and highlight the importance of FBXO38 and KLF7 activity in motor neurons.

Charcot-Marie-Tooth disease: emerging mechanisms and therapies.

Charcot-Marie-Tooth disease is the most common inherited disorder of the peripheral nervous system. The disease is characterized by a progressive muscle weakness and atrophy, sensory loss, foot (and hand) deformities and steppage gait. While many of the genes associated with axonal CMT have been identified, to date it is unknown which mechanism(s) causes the disease. However, genetic findings indicate that the underlying mechanisms mainly converge to the axonal cytoskeleton. In this review, we will summarize the evidence for this pathogenic convergence. Furthermore, recent work with new transgenic mouse models has led to the identification of histone deacetylase 6 as a potential therapeutic target for inherited peripheral neuropathies. This enzyme deacetylates microtubules and plays a crucial role in the regulation of axonal transport. These findings offer new perspectives for a potential therapy to treat axonal Charcot-Marie-Tooth disease and other neurodegenerative disorders characterized by axonal transport defects.

HDAC6 at the Intersection of Neuroprotection and Neurodegeneration.

Histone deacetylase 6 (HDAC6) catalyzes multiple reactions. We summarize the current knowledge on HDAC6, its targets and functions. Among others, HDAC6 recognizes damaged proteins and assures that these proteins are destroyed by autophagy. On the other hand, HDAC6 also modifies the tracks used by the clearance mechanism so that axonal transport becomes less efficient. We hypothesize that a disturbance in the equilibrium between the different functions of HDAC6 could play an important role in neurodegeneration.

Small heat-shock protein HSPB1 mutants stabilize microtubules in Charcot-Marie-Tooth neuropathy.

Mutations in the small heat shock protein HSPB1 (HSP27) are causative for Charcot-Marie-Tooth (CMT) neuropathy. We previously showed that a subset of these mutations displays higher chaperone activity and enhanced affinity to client proteins. We hypothesized that this excessive binding property might cause the HSPB1 mutant proteins to disturb the function of proteins essential for the maintenance or survival of peripheral neurons. In the present work, we explored this hypothesis further and compared the protein complexes formed by wild-type and mutant HSPB1. Tubulin came out as the most striking differential interacting protein, with hyperactive mutants binding more strongly to both tubulin and microtubules. This anomalous binding leads to a stabilization of the microtubule network in a microtubule-associated protein-like manner as reflected by resistance to cold depolymerization, faster network recovery after nocodazole treatment, and decreased rescue and catastrophe rates of individual microtubules. In a transgenic mouse model for mutant HSPB1 that recapitulates all features of CMT, we could confirm the enhanced interaction of mutant HSPB1 with tubulin. Increased stability of the microtubule network was also clear in neurons isolated from these mice. Since neuronal cells are particularly vulnerable to disturbances in microtubule dynamics, this mechanism might explain the neuron-specific CMT phenotype caused by HSPB1 mutations.

HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common inherited disorder of the peripheral nervous system. Mutations in the 27-kDa small heat-shock protein gene (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). We developed and characterized transgenic mice expressing two different HSPB1 mutations (S135F and P182L) in neurons only. These mice showed all features of CMT or distal HMN dependent on the mutation. Expression of mutant HSPB1 decreased acetylated α-tubulin abundance and induced severe axonal transport deficits. An increase of α-tubulin acetylation induced by pharmacological inhibition of histone deacetylase 6 (HDAC6) corrected the axonal transport defects caused by HSPB1 mutations and rescued the CMT phenotype of symptomatic mutant HSPB1 mice. Our findings demonstrate the pathogenic role of α-tubulin deacetylation in mutant HSPB1-induced neuropathies and offer perspectives for using HDAC6 inhibitors as a therapeutic strategy for hereditary axonopathies.

Effects of mental stress on autonomic cardiac modulation during weightlessness.

Sustained weightlessness affects all body functions, among these also cardiac autonomic control mechanisms. How this may influence neural response to central stimulation by a mental arithmetic task remains an open question. The hypothesis was tested that microgravity alters cardiovascular neural response to standardized cognitive load stimuli. Beat-to-beat heart rate, brachial blood pressure, and respiratory frequency were collected in five astronauts, taking part in three different short-duration (10 to 11 days) space missions to the International Space Station. Data recording was performed in supine position 1 mo before launch; at days 5 or 8 in space; and on days 1, 4, and 25 after landing. Heart rate variability (HRV) parameters were obtained in the frequency domain. Measurements were performed in the control condition for 10 min and during a 5-min mental arithmetic stress task, consisting of deducting 17 from a four-digit number, read by a colleague, and orally announcing the result. Our results show that over all sessions (pre-, in-, and postflight), mental stress induced an average increase in mean heart rate (Delta7 +/- 1 beats/min; P = 0.03) and mean arterial pressure (Delta7 +/- 1 mmHg; P = 0.006). A sympathetic excitation during mental stress was shown from HRV parameters: increase of low frequency expressed in normalized units (Delta8.3 +/- 1.4; P = 0.004) and low frequency/high frequency (Delta1.6 +/- 0.3; P = 0.001) and decrease of high frequency expressed in normalized units (Delta8.9 +/- 1.4; P = 0.004). The total power was not influenced by mental stress. No effect of spaceflight was found on baseline heart rate, mean arterial pressure, and HRV parameters. No differences in response to mental stress were found between pre-, in-, and postflight. Our findings confirm that a mental arithmetic task in astronauts elicits sympathovagal shifts toward enhanced sympathetic modulation and reduced vagal modulation. However, these responses are not changed in space during microgravity or after spaceflight.