RNA interference (RNAi)-based therapeutics for treatment of rare neurologic diseases.

Advances in genome sequencing have greatly facilitated the identification of genomic variants underlying rare neurodevelopmental and neurodegenerative disorders. Understanding the fundamental causes of rare monogenic disorders has made gene therapy a possible treatment approach for these conditions. RNA interference (RNAi) technologies such as small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA), and other oligonucleotide-based modalities such as antisense oligonucleotides (ASOs) are being developed as potential therapeutic approaches for manipulating expression of the genes that cause a variety of neurological diseases. Here, we offer a brief review of the mechanism of action of these RNAi approaches; provide deeper discussion of the advantages, challenges, and specific considerations related to the development of RNAi therapeutics for neurological disease; and highlight examples of rare neurological diseases for which RNAi therapeutics hold great promise.

The Evaluation of Magnesium Chloride within a Polyethylene Glycol Formulation in a Porcine Model of Acute Spinal Cord Injury.

A porcine model of spinal cord injury (SCI) was used to evaluate the neuroprotective effects of magnesium chloride (MgCl2) within a polyethylene glycol (PEG) formulation, called "AC105" (Acorda Therapeutics Inc., Ardsley, NY). Specifically, we tested the hypothesis that AC105 would lead to greater tissue sparing at the injury site and improved behavioral outcome when delivered in a clinically realistic time window post-injury. Four hours after contusion/compression injury, Yucatan minipigs were randomized to receive a 30-min intravenous infusion of AC105, magnesium sulfate (MgSO4), or saline. Animals received 4 additional infusions of the same dose at 6-h intervals. Behavioral recovery was tested for 12 weeks using two-dimensional (2D) kinematics during weight-supported treadmill walking and the Porcine Injury Behavior Scale (PTIBS), a 10-point locomotion scale. Spinal cords were evaluated ex vivo by diffusion-weighted magnetic resonance imaging (MRI) and subjected to histological analysis. Treatment with AC105 or MgSO4 did not result in improvements in locomotor recovery on the PTIBS or in 2D kinematics on weight-supported treadmill walking. Diffusion weighted imaging (DWI) showed severe loss of tissue integrity at the impact site, with decreased fractional anisotropy and increased mean diffusivity; this was not improved with AC105 or MgSO4 treatment. Histological analysis revealed no significant increase in gray or white matter sparing with AC105 or MgSO4 treatment. Finally, AC105 did not result in higher Mg2+ levels in CSF than with the use of standard MgSO4. In summary, when testing AC105 in a porcine model of SCI, we were unable to reproduce the promising therapeutic benefits observed previously in less-severe rodent models of SCI.

The Kv2.1 K+ channel targets to the axon initial segment of hippocampal and cortical neurons in culture and in situ.

BACKGROUND: The Kv2.1 delayed-rectifier K+ channel regulates membrane excitability in hippocampal neurons where it targets to dynamic cell surface clusters on the soma and proximal dendrites. In the past, Kv2.1 has been assumed to be absent from the axon initial segment. RESULTS: Transfected and endogenous Kv2.1 is now demonstrated to preferentially accumulate within the axon initial segment (AIS) over other neurite processes; 87% of 14 DIV hippocampal neurons show endogenous channel concentrated at the AIS relative to the soma and proximal dendrites. In contrast to the localization observed in pyramidal cells, GAD positive inhibitory neurons within the hippocampal cultures did not show AIS targeting. Photoactivable-GFP-Kv2.1-containing clusters at the AIS were stable, moving <1 microm/hr with no channel turnover. Photobleach studies indicated individual channels within the cluster perimeter were highly mobile (FRAP tau=10.4+/-4.8 sec), supporting our model that Kv2.1 clusters are formed by the retention of mobile channels behind a diffusion-limiting perimeter. Demonstrating that the AIS targeting is not a tissue culture artifact, Kv2.1 was found in axon initial segments within both the adult rat hippocampal CA1, CA2, and CA3 layers and cortex. CONCLUSION: In summary, Kv2.1 is associated with the axon initial segment both in vitro and in vivo where it may modulate action potential frequency and back propagation. Since transfected Kv2.1 initially localizes to the AIS before appearing on the soma, it is likely multiple mechanisms regulate Kv2.1 trafficking to the cell surface.

Statins decrease chondroitin sulfate proteoglycan expression and acute astrocyte activation in central nervous system injury.

Statins elicit numerous favorable effects on central nervous system (CNS) injury, including inhibition of the rhoA/ROCK pathway. In the present study, we show that statins decrease acute astrocyte activation in CNS injury, and decrease chondroitin sulfate proteoglycan (CSPG) levels in astrocyte cultures as well as CNS injury. CSPG levels decreased by up to 45% in simvastatin-treated astrocyte cultures compared to control cultures. In simvastatin-treated animals, CSPG levels declined by 60% 8 days after brain stab injury, and by 62-64% 4 weeks after spinal cord injury (SCI). Glial fibrillary acid protein (GFAP) levels decreased in brain stab at 8 days after surgery/intervention, suggesting that statins produce a decrease in astrocyte activation. Attenuation of astrocyte activation may contribute to the decline in CSPG levels. However, there are likely other contributing factors, since GFAP levels were not a contributing factor in the decline of CSPG levels in astrocyte cultures. Robust locomotor improvements were not observed with any treatment. The numerous beneficial effects of statins on CNS injury render them an attractive candidate in the treatment of CNS injury.

Brain-derived neurotrophic factor regulation of retinal growth cone filopodial dynamics is mediated through actin depolymerizing factor/cofilin.

The molecular mechanisms by which neurotrophins regulate growth cone motility are not well understood. This study investigated the signaling involved in transducing BDNF-induced increases of filopodial dynamics. Our results indicate that BDNF regulates filopodial length and number through a Rho kinase-dependent mechanism. Additionally, actin depolymerizing factor (ADF)/cofilin activity is necessary and sufficient to transduce the effects of BDNF. Our data indicate that activation of ADF/cofilin mimics the effects of BDNF on filopodial dynamics, whereas ADF/cofilin inactivity blocks the effects of BDNF. Furthermore, BDNF promotes the activation of ADF/cofilin by reducing the phosphorylation of ADF/cofilin. Although inhibition of myosin II also enhances filopodial length, our results indicate that BDNF signaling is independent of myosin II activity and that the two pathways result in additive effects on filopodial length. Thus, filopodial extension is regulated by at least two independent mechanisms. The BDNF-dependent pathway works via regulation of ADF/cofilin, independently of myosin II activity.

Efficient Salmonella entry requires activity cycles of host ADF and cofilin.

Entry of Salmonella into mammalian cells is strictly dependent on the reorganization of actin cytoskeleton induced by a panel of Salmonella type III secreted proteins. Although several factors have been identified to be responsible for inducing the actin polymerization and stability, little is known about how the actin depolymerization contributes to Salmonella-induced actin rearrangements. We report here that activity cycles of host actin depolymerizing factor (ADF and cofilin) are modulated by Salmonella during bacterial entry. Efficient Salmonella internalization involves an initial dephosphorylation of ADF and cofilin followed by phosphorylation, suggesting that ADF and cofilin activities are increased briefly. Expression of a kinase dead form of an ADF/cofilin kinase (LIM kinase 1) or a catalytically inactive ADF/cofilin phosphatase (Slingshot), but not constitutively active LIM kinase 1 or wild-type Slingshot, resulted in decreased invasion. These data suggest that ADF/cofilin activities play a key role in the actin polymerization/depolymerization process induced by Salmonella. The activation of ADF/cofilin is brief and has to be reversed to facilitate efficient bacterial entry. Surprisingly, co-expression of constitutive active ADF and cofilin prevented efficient Salmonella entry, whereas expression of either one alone had no effect. We propose that ADF and cofilin actin-dynamizing activities and their activity cycling via phosphorylation are required for efficient Salmonella internalization.

LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues.

The expression of endogenous LIM kinase 1 (LIMK1) protein was investigated in embryonic and adult mice using a rat monoclonal antibody (mAb), which recognizes specifically the PDZ domain of LIMK1 and not LIMK2. Immunoblotting analysis revealed widespread expression of LIMK1 existing as a 70-kDa protein in tissues and in cell lines, with a higher mass form (approximately 75 kDa) present in some tissues and cell lines. Smaller isoforms of approximately 50 kDa were also occasionally evident. Immunofluorescence analysis demonstrated LIMK1 subcellular localization at focal adhesions in fibroblasts as revealed by co-staining with actin, paxillin and vinculin in addition to perinuclear (Golgi) and occasional nuclear localization. Furthermore, an association between LIMK1 and paxillin but not vinculin was identified by co-immunoprecipitation analysis. LIMK1 is enriched in both axonal and dendritic growth cones of E18 rat hippocampal pyramidal neurons where it is found in punctae that extend far out into filopodia, as well as in a perinuclear region identified as Golgi. In situ, we identify LIMK1 protein expression in all embryonic and adult tissues examined, albeit at different levels and in different cell populations. The rat monoclonal LIMK1 antibody recognizes proteins of similar size in cell and tissue extracts from numerous species. Thus, LIMK1 is a widely expressed protein that exists as several isoforms.

Regulation of the neuronal actin cytoskeleton by ADF/cofilin.

Actin and microtubules are major cytoskeletal elements of most cells including neurons. In order for a cell to move and change shape, its cytoskeleton must undergo rearrangements that involve breaking down and reforming filaments. Many recent reviews have focused on the signaling pathways emanating from receptors that ultimately affect axon growth and growth cone steering. This particular review will address changes in the actin cytoskeleton modulated by the family of actin dynamizing proteins known as actin depolymerizing factor (ADF)/cofilin or AC proteins. Though much is known about inactivation of AC proteins through phosphorylation at ser3 by LIM or TES kinases, new mechanisms of regulation of AC have recently emerged. A novel phosphatase, slingshot (SSH), and the 14-3-3 family of regulatory proteins have also been found to affect AC activity. The potential role of AC proteins in modulating the actin organizational changes that accompany neurite initiation, axonogenesis, growth cone guidance, and dendritic spine formation will be discussed.

Head, neck, and spines: a role for LIMK-1 in the hippocampus.

LIM kinase 1 regulates actin filament dynamics through inhibition of ADF/cofilins. Surprisingly, nervous system development in LIM kinase 1 knockout mice is grossly normal, but the animals have deficits in spatial learning, alterations in LTP, and abnormalities in hippocampal dendritic spine structure. The findings are consistent with a role for LIMK-1 in synapse formation and function.