Molecular genetic investigations of histone deacetylase inhibitors as potential neurotherapeutics for autosomal recessive proximal spinal muscular atrophy (SMA)

Proximal spinal muscular atrophy (SMA) is a common neuromuscular disorder causing infant death in 50 percent of all patients. Homozygous absence of the survival motor neuron gene (SMN1) is the primary cause of SMA, while SMA severity is mainly determined by the number of SMN2 copies. One SMN2 copy produces only about 10 percent of full-length (FL) protein identical to SMN1, whereas the majority of SMN2 transcripts are aberrantly spliced due to a silent mutation within an exonic splicing enhancer in exon 7. However, correct splicing can be restored by over-expression of the SR-like splicing factor Htra2-beta1. In the present work, it is demonstrated that in fibroblast cultures derived from SMA patients treated with therapeutic doses (0.5-50 microM) of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) the level of SMN protein increased about 3fold. Augmented SMN protein levels could be attributed to elevated FL-SMN2 transcript levels which were triggered by two different mechanisms: a transcriptional activation of the SMN2 gene, and a preferred exon 7 inclusion in SMN2 transcripts. The latter observation was most likely due to increased levels of the splicing factor Htra2-beta1. In addition to Htra2-beta1, VPA increased the expression of further SR proteins which may have important implications for other disorders affected by alternative splicing. Importantly, the drug was able to elevate rSmn transcript and protein levels ex vivo through transcriptional activation in organotypic hippocampal brain slices from rats. This demonstrated that VPA also exerts an effect on neuronal tissue, the target for a potential SMA therapy. Since VPA is a drug highly successfully used in long-time epilepsy therapy, these findings opened the exciting perspective for a first causal therapy of an inherited disease by elevating the SMN2 transcription level and restoring its correct splicing. The evaluation of two second-generation HDAC inhibitors in SMA fibroblasts in vitro revealed that SAHA, a drug that belongs to the hydroxamic acids, also efficiently elevated SMN protein levels 2.4fold to 3fold. Therefore, SAHA was identified as another attractive candidate for SMA therapy. In contrast, the data obtained for MS-275, an HDAC inhibitor of the benzamide class, demonstrated that the drug does not possess enough potency to substantially elevate SMN protein levels in vitro. Thus, MS-275 will not have a chance to move forward to SMA clinical trials. Based on the promising data for VPA in vitro and ex vivo and given that VPA is already approved for application to humans, a first pilot trial with VPA was carried out in ten SMA carriers (parents of patients with SMA) aiming to evaluate drug potency to increase SMN transcript and protein levels in vivo. In order to further validate the outcome of the study, SMN2 gene expression was analyzed in peripheral whole blood derived from 20 patients with SMA (5x type I, 11x type II, 4x type III) treated with VPA in individual experimental curative approaches all over Germany according to section 41 of the German Drug Act (AMG). Moreover, the value of these screenings was determined for the development of a clinical biomarker to monitor the response to VPA and other HDAC inhibitors in treated individuals. Such a biomarker would be highly useful for clinical trials and future therapies in SMA patients. Drug treatment revealed elevated full-length SMN RNA and protein levels in blood from 7/10 SMA carriers. Importantly, SMN protein levels increased far more substantially (up to 13.8fold) than the levels of the intermediate product, FL-SMN RNA, that showed an increase of up to 3.4fold. These observations provided first proof of the in vivo activation of a therapeutic target gene by VPA in an inherited disease. Among the investigated SMA patients, FL-SMN2 RNA levels were increased 1.5fold to 1.9fold in seven subjects, whereas 13 patients presented unchanged or even decreased transcript levels. This data suggested that some individuals are responders to VPA, while others are most likely nonresponders or even negative-responders. However, so far it is unknown whether SMN expression in blood reflects SMN expression in alpha-motor neurons and correlates with muscle strength. Therefore, systematic long-term clinical trials in SMA patients that correlate SMN expression in blood with individual motor function tests are required in the future to address the question whether SMN transcript and protein levels in blood may serve as biomarker, and to study the effect of VPA on motor function

An ∼140-kb Deletion Associated with Feline Spinal Muscular Atrophy Implies an Essential LIX1 Function for Motor Neuron Survival

The leading genetic cause of infant mortality is spinal muscular atrophy (SMA), a clinically and genetically heterogeneous group of disorders. Previously we described a domestic cat model of autosomal recessive, juvenile-onset SMA similar to human SMA type III. Here we report results of a whole-genome scan for linkage in the feline SMA pedigree using recently developed species-specific and comparative mapping resources. We identified a novel SMA gene candidate, LIX1, in an ~140-kb deletion on feline chromosome A1q in a region of conserved synteny to human chromosome 5q15. Though LIX1 function is unknown, the predicted secondary structure is compatible with a role in RNA metabolism. LIX1 expression is largely restricted to the central nervous system, primarily in spinal motor neurons, thus offering explanation of the tissue restriction of pathology in feline SMA. An exon sequence screen of 25 human SMA cases, not otherwise explicable by mutations at the SMN1 locus, failed to identify comparable LIX1 mutations. Nonetheless, a LIX1-associated etiology in feline SMA implicates a previously undetected mechanism of motor neuron maintenance and mandates consideration of LIX1 as a candidate gene in human SMA when SMN1 mutations are not found

Transient Activation of GABA_B Receptors Suppresses SK Channel Currents in Substantia Nigra Pars Compacta Dopaminergic Neurons

<div><p>Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) are richly innervated by GABAergic neurons. The postsynaptic effects of GABA on SNc DA neurons are mediated by a mixture of GABA_A and GABA_B receptors. Although activation of GABA_A receptors inhibits spike generation, the consequences of GABA_B receptor activation are less well characterized. To help fill this gap, perforated patch recordings were made from young adult mouse SNc DA neurons. Sustained stimulation of GABA_B receptors hyperpolarized SNc DA neurons, as previously described. However, transient stimulation of GABA_B receptors by optical uncaging of GABA did not; rather, it reduced the opening of small-conductance, calcium-activated K⁺ (SK) channels and increased the irregularity of spiking. This modulation was attributable to inhibition of adenylyl cyclase and protein kinase A. Thus, because suppression of SK channel activity increases the probability of burst spiking, transient co-activation of GABA_A and GABA_B receptors could promote a pause-burst pattern of spiking.</p></div

VGCC contribution to SK.

<p>(A) Application of 10 μM isradipine (orange) to inhibit Ca_V1 channels did not reduce total SK charge (n = 8, Wilcoxon signed rank test, p = 0.3828), while inhibiting Ca_V3 channels with 10 μM mibefradil (blue) inhibited roughly half the charge (n = 8, Wilcoxon signed rank test, p = 0.0078). (B) 5 μM baclofen (green) application did not inhibit T-type calcium current (n = 12, Wilcoxon signed rank test, p = 0.1099).</p

PKA activation prevents GABA_B modulation of SK.

<p>(A) Schematic diagram showing the hypothesized signaling pathway from GABA_B receptor activation to SK channels, and the site of action of 8-bromo-cAMP and H-89 in that pathway. (B) A 50 ms uncaging pulse elicited an immediate and significant change in SOP variance (black, n = 10, Wilcoxon signed rank test, p = 0.002). (C) The same was not seen when cells were incubated in 1 μM 8-bromo-cAMP (orange, n = 4, Wilcoxon signed rank test, p = 0.875). (D) Directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SOP variance (n = 4, Wilcoxon signed rank test, p = 0.75). (E) Inhibiting PKA with 10 μM H89 increases SOP variance (n = 6, Wilcoxon signed rank test, p = 0.0938). (F) Summary data for panels A-B. (G) Summary data for panels C-D. (H) Top, inhibiting PKA with 10 μM H89 significantly decreases SK current (n = 6, Wilcoxon signed rank test, p = 0.0313). Bottom, directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SK current (n = 5, Wilcoxon signed rank test, p = 1.00). (I) Summary data for PKA modulators from panel H and Rp-8-CPT-cAMPS (n = 3, Wilcoxon signed rank test, p = 0.25).</p

RuBi-GABA uncaging.

<p>(A) Top, plot of the normalized firing rate before during and after a 60 s uncaging pulse (blue bar) of 5 μM RuBi-GABA in the presence of 25 μM gabazine (n = 4). Bottom, plot of normalized firing rate (black line) and running standard deviation (grey area) before, during and after a 50 ms uncaging pulse in the presence of 5 μM RuBi-GABA and 25 μM gabazine (n = 12); application of 2 μM CGP 55845 blunted the changes in spiking induced by RuBi-GABA uncaging (orange line, n = 4). Example raster plots are shown at the top of the panel. (B) Left, two different time scales showing action potentials just prior to and after GABA uncaging. Right, overlaid action potentials from just prior to and after GABA uncaging showing a clear reduction in the mAHP. (C-D) As in panel A (bottom) and B, but in the absence of gabazine (n = 9).</p

SNc DA neuron physiology.

<p>(A) 2P reconstruction of SNc DA neuron. (B) Left, normal pacemaking of a SNc DA neuron. Middle, TTX (1 μM) application uncovered slow oscillatory potential (SOP). Right, 5 μM baclofen application hyperpolarized the cell. (C) Summary of hyperpolarization due to application of 5 μM baclofen (n = 8, median = -25.24 mV). (D) Sustained uncaging of 5 μM RuBi-GABA in the presence of 25 μM gabazine (to block GABA_A receptors) hyperpolarized cells in a manner similar to that seen following baclofen application. (E) Summary of hyperpolarization due to sustained 5 μM RuBi-GABA uncaging (n = 7, median = -11.71 mV).</p

Schematic diagram depicting hypothesized signaling pathways involved in the GABA_B receptor-mediated inhibition of SK channels.

<p>GABA_B receptor inhibition of AC by G_i signaling is hypothesized to be responsible for reduced cAMP levels and PKA signaling. The reduction in PKA activity is hypothesized to reduce SK channel opening through mechanism that are independent of either plasma membrane Ca²⁺ channels or release from intracellular stores.</p

Author Correction: Dopamine metabolism by a monoamine oxidase mitochondrial shuttle activates the electron transport chain

An amendment to this paper has been published and can be accessed via a link at the top of the paper