Emerging siRNA Design Principles and Consequences for Biotransformation and Disposition in Drug Development.

After two decades teetering at the intersection of laboratory tool and therapeutic reality, with two siRNA drugs now clinically approved, this modality has finally come into fruition. Consistent with other emerging modalities, initial proof-of-concept efforts concentrated on coupling pharmacologic efficacy with desirable safety profiles. Consequently, thorough investigations of siRNA absorption, distribution, metabolism, and excretion (ADME) properties are lacking.

Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss.

Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice.

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5.

The C-terminal domains of NF-H and NF-M subunits maintain axonal neurofilament content by blocking turnover of the stationary neurofilament network.

Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)(tailΔ)] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF.

The myosin Va head domain binds to the neurofilament-L rod and modulates endoplasmic reticulum (ER) content and distribution within axons.

The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon.

Marked calpastatin (CAST) depletion in Alzheimer's disease accelerates cytoskeleton disruption and neurodegeneration: neuroprotection by CAST overexpression.

Increased activity of calpains is implicated in synaptic dysfunction and neurodegeneration in Alzheimer's disease (AD). The molecular mechanisms responsible for increased calpain activity in AD are not known. Here, we demonstrate that disease progression is propelled by a marked depletion of the endogenous calpain inhibitor, calpastatin (CAST), from AD neurons, which is mediated by caspase-1, caspase-3, and calpains.

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate.

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V.