Kenneth J. Rhodes

The antibody aducanumab reduces Aβ plaques in Alzheimer's disease.

Alzheimer’s disease (AD) is characterized by deposition of amyloid-β (Aβ) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based immunotherapy against Aβ to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated Aβ. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aβ, and reduce soluble and insoluble Aβ in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aβ in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating—Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

Fumarates Promote Cytoprotection of Central Nervous System Cells against Oxidative Stress via the Nuclear Factor (Erythroid-Derived 2)-Like 2 Pathway

Oxidative stress is central to the pathology of several neurodegenerative diseases, including multiple sclerosis, and therapeutics designed to enhance antioxidant potential could have clinical value. The objective of this study was to characterize the potential direct neuroprotective effects of dimethyl fumarate (DMF) and its primary metabolite monomethyl fumarate (MMF) on cellular resistance to oxidative damage in primary cultures of central nervous system (CNS) cells and further explore the dependence and function of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway in this process. Treatment of animals or primary cultures of CNS cells with DMF or MMF resulted in increased nuclear levels of active Nrf2, with subsequent up-regulation of canonical antioxidant target genes. DMF-dependent up-regulation of antioxidant genes in vivo was lost in mice lacking Nrf2 [Nrf2(−/−)]. DMF or MMF treatment increased cellular redox potential, glutathione, ATP levels, and mitochondrial membrane potential in a concentration-dependent manner. Treating astrocytes or neurons with DMF or MMF also significantly improved cell viability after toxic oxidative challenge in a concentration-dependent manner. This effect on viability was lost in cells that had eliminated or reduced Nrf2. These data suggest that DMF and MMF are cytoprotective for neurons and astrocytes against oxidative stress-induced cellular injury and loss, potentially via up-regulation of an Nrf2-dependent antioxidant response. These data also suggest DMF and MMF may function through improving mitochondrial function. The clinical utility of DMF in multiple sclerosis is being explored through phase III trials with BG-12, which is an oral therapeutic containing DMF as the active ingredient.

KChIPs and Kv4 α Subunits as Integral Components of A-Type Potassium Channels in Mammalian Brain

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family α subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family α subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 α subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain.

The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 α-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1–3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific “ball” domain of the Kvβ1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.

Extension of the Thrombolytic Time Window With Minocycline in Experimental Stroke

Background and Purpose— Thrombolysis with tPA is the only FDA-approved therapy for acute ischemic stroke. But its widespread application remains limited by narrow treatment time windows and the related risks of cerebral hemorrhage. In this study, we ask whether minocycline can prevent tPA-associated cerebral hemorrhage and extend the reperfusion window in an experimental stroke model in rats. Methods— Spontaneously hypertensive rats were subjected to embolic focal ischemia using homologous clots and treated with: saline at 1 hour; early tPA at 1 hour, delayed tPA at 6 hours; minocycline at 4 hours; combined minocycline at 4 hours plus tPA at 6 hours. Infarct volumes and hemorrhagic transformation were quantified at 24 hours. Gelatin zymography was used to measure blood levels of circulating matrix metalloproteinase-9 (MMP-9). Results— Early 1-hour thrombolysis restored perfusion and reduced infarction. Late 6-hour tPA did not decrease infarction but instead worsened hemorrhagic conversion. Combining minocycline with delayed 6-hour tPA decreased plasma MMP-9 levels, reduced infarction, and ameliorated brain hemorrhage. Blood levels of MMP-9 were also significantly correlated with volumes of infarction and hemorrhage. Conclusion— Combination therapy with minocycline may extend tPA treatment time windows in ischemic stroke.

Immunohistochemical localization of subtype 4a metabotropic glutamate receptors in the rat and mouse basal ganglia

Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild‐type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a‐transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g‐amino‐n‐butyric acid (GABA) release from striatopallidal terminals. J. Comp. Neurol. 407:33–46, 1999.

Role of Heteromultimers in the Generation of Myocardial Transient Outward K+ Currents

Previous studies have demonstrated a role for Kv4 &agr; subunits in the generation of the fast transient outward K+ current, Ito,f, in the mammalian myocardium. The experiments here were undertaken to explore the role of homomeric/heteromeric assembly of Kv4.2 and Kv4.3 and of the Kv channel accessory subunit, KChIP2, in the generation of mouse ventricular Ito,f. Western blots reveal that the expression of Kv4.2 parallels the regional heterogeneity in Ito,f density, whereas Kv4.3 and KChIP2 are uniformly expressed in adult mouse ventricles. Antisense oligodeoxynucleotides (AsODNs) targeted against Kv4.2 or Kv4.3 selectively attenuate Ito,f in mouse ventricular cells. Adenoviral-mediated coexpression of Kv4.2 and Kv4.3 in HEK-293 cells and in mouse ventricular myocytes produces transient outward K+ currents with properties distinct from those produced on expression of Kv4.2 or Kv4.3 alone, and the gating properties of the heteromeric Kv4.2/Kv4.3 channels in ventricular cells are more similar to native Ito,f than are the homomeric Kv4.2 or Kv4.3 channels. Biochemical studies reveal that Kv4.2, Kv4.3, and KChIP2 coimmunoprecipitate from adult mouse ventricles. In addition, most of the Kv4.2 and KChIP2 are associated with Kv4.3 in situ. Taken together, these results demonstrate that functional mouse ventricular Ito,f channels are heteromeric, comprising Kv4.2/Kv4.3 &agr; subunits and KChIP2. The results here also suggest that Kv4.2 is the primary determinant of the regional heterogeneity in Ito,f expression in adult mouse ventricle.

Antibodies as Valuable Neuroscience Research Tools versus Reagents of Mass Distraction

Editors Note: Toolboxes are a new, occasional feature in the Journal designed to briefly highlight a new method or a resource of general use in neuroscience or to critically analyze existing approaches or methods. For more information, see [http://www.jneurosci.org/misc/sifa_features.shtml][1].

Remodelling inactivation gating of Kv4 channels by KChIP1, a small-molecular-weight calcium-binding protein

Calcium‐binding proteins dubbed KChIPs favour surface expression and modulate inactivation gating of neuronal and cardiac A‐type Kv4 channels. To investigate their mechanism of action, Kv4.1 or Kv4.3 were expressed in Xenopus laevis oocytes, either alone or together with KChIP1, and the K+ currents were recorded using the whole‐oocyte voltage‐clamp and patch‐clamp methods. KChIP1 similarly remodels gating of both channels. At positive voltages, KChIP1 slows the early phase of the development of macroscopic inactivation. By contrast, the late phase is accelerated, which allows complete inactivation in < 500 ms. Thus, superimposed traces from control and KChIP1‐remodelled currents crossover. KChIP1 also accelerates closed‐state inactivation and recovery from inactivation (3‐ to 5‐fold change). The latter effect is dominating and, consequently, the prepulse inactivation curves exhibit depolarizing shifts (ΔV= 4–12 mV). More favourable closed‐state inactivation may also contribute to the overall faster inactivation at positive voltages because Kv4 channels significantly inactivate from the preopen closed state. KChIP1 favours this pathway further by accelerating channel closing. The peak G‐V curves are modestly leftward shifted in the presence of KChIP1, but the apparent ‘threshold’ voltage of current activation remains unaltered. Single Kv4.1 channels exhibited multiple conductance levels that ranged between 1.8 and 5.6 pS in the absence of KChIP1 and between 1.9 and 5.3 pS in its presence. Thus, changes in unitary conductance do not contribute to current upregulation by KChIP1. An allosteric kinetic model explains the kinetic changes by assuming that KChIP1 mainly impairs open‐state inactivation, favours channel closing and lowers the energy barrier of closed‐state inactivation.