Excitotoxic mechanisms contribute to the degeneration of hippocampal pyramidal neurons after recurrent seizures and brain ischemia. However, susceptibility differs, with CA1 neurons degenerating preferentially after global ischemia and CA3 neurons after limbic seizures. Whereas most studies address contributions of excitotoxic Ca²⁺ entry, it is apparent that Zn²⁺ also contributes, reflecting accumulation in neurons either after synaptic release and entry through postsynaptic channels or upon mobilization from intracellular Zn²⁺-binding proteins such as metallothionein-III (MT-III). Using mouse hippocampal slices to study acute oxygen glucose deprivation (OGD)-triggered neurodegeneration, we found evidence for early contributions of excitotoxic Ca²⁺ and Zn²⁺ accumulation in both CA1 and CA3, as indicated by the ability of Zn²⁺ chelators or Ca²⁺ entry blockers to delay pyramidal neuronal death in both regions. However, using knock-out animals (of MT-III and vesicular Zn²⁺ transporter, ZnT3) and channel blockers revealed substantial differences in relevant Zn²⁺ sources, with critical contributions of presynaptic release and its permeation through Ca²⁺- (and Zn²⁺)-permeable AMPA channels in CA3 and Zn²⁺ mobilization from MT-III predominating in CA1. To assess the consequences of the intracellular Zn²⁺ accumulation, we used OGD exposures slightly shorter than those causing acute neuronal death; under these conditions, cytosolic Zn²⁺ rises persisted for 10-30 min after OGD, followed by recovery over ∼40-60 min. Furthermore, the recovery appeared to be accompanied by mitochondrial Zn²⁺ accumulation (via the mitochondrial Ca²⁺ uniporter MCU) in CA1 but not in CA3 neurons and was markedly diminished in MT-III knock-outs, suggesting that it depended upon Zn²⁺ mobilization from this protein.

Significance statement

The basis for the differential vulnerabilities of CA1 versus CA3 pyramidal neurons is unclear. The present study of events during and after acute oxygen glucose deprivation highlights a possible important difference, with rapid synaptic entry of Ca²⁺ and Zn²⁺ contributing more in CA3, but with delayed and long-lasting accumulation of Zn²⁺ within mitochondria occurring in CA1 but not CA3 pyramidal neurons. These data may be consistent with observations of prominent mitochondrial dysfunction as a critical early event in the delayed degeneration of CA1 neurons after ischemia and support a hypothesis that mitochondrial Zn²⁺ accumulation in the early reperfusion period may be a critical and targetable upstream event in the injury cascade.

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca²⁺ overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn²⁺, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this review, we discuss both old and new findings yielding clues as to sources of the Zn²⁺ that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury. Specifically, we highlight the growing evidence of important Zn²⁺ effects on mitochondria in promoting neuronal injury. A key focus has been to examine Zn²⁺ contributions to the degeneration of highly susceptible hippocampal pyramidal neurons. Recent studies provide evidence of differences in sources of Zn²⁺ and its interactions with mitochondria in CA1 versus CA3 neurons that may pertain to their differential vulnerabilities in disease. We propose that Zn²⁺-induced mitochondrial dysfunction is a critical and potentially targetable early event in the ischemic neuronal injury cascade, providing opportunities for the development of novel neuroprotective strategies to be delivered after transient ischemia.

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca²⁺ overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn²⁺, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this review, we discuss both old and new findings yielding clues as to sources of the Zn²⁺ that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury. Specifically, we highlight the growing evidence of important Zn²⁺ effects on mitochondria in promoting neuronal injury. A key focus has been to examine Zn²⁺ contributions to the degeneration of highly susceptible hippocampal pyramidal neurons. Recent studies provide evidence of differences in sources of Zn²⁺ and its interactions with mitochondria in CA1 versus CA3 neurons that may pertain to their differential vulnerabilities in disease. We propose that Zn²⁺-induced mitochondrial dysfunction is a critical and potentially targetable early event in the ischemic neuronal injury cascade, providing opportunities for the development of novel neuroprotective strategies to be delivered after transient ischemia.

Epilepsy may result from abnormal function of ion channels, such as those caused by genetic mutations. Recently, pathological alterations of the expression or localization of normal channels have been implicated in epilepsy generation, and termed 'acquired channelopathies'. Altered expression levels of the HCN channels - that conduct the hyperpolarization-activated current, I(h) - have been demonstrated in hippocampus of patients with severe temporal lobe epilepsy as well as in animal models of temporal lobe and absence epilepsies. Here we probe the mechanisms for the altered expression of HCN channels which is provoked by seizures. In organotypic hippocampal slice cultures, seizure-like events selectively reduced HCN type 1 channel expression and increased HCN2 mRNA levels, as occurs in vivo. The mechanisms for HCN1 reduction involved Ca(2+)-permeable AMPA receptor-mediated Ca(2+) influx, and subsequent activation of Ca(2+)/calmodulin-dependent protein kinase II. In contrast, upregulation of HCN2 expression was independent of these processes. The data demonstrate an orchestrated program for seizure-evoked transcriptional channelopathy involving the HCN channels that may contribute to certain epilepsies.

Mitochondrial Zn2+ accumulation, particularly in CA1 neurons, occurs after ischemia and likely contributes to mitochondrial dysfunction and subsequent neurodegeneration. However, the relationship between mitochondrial Zn2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo. We employed a cardiac arrest model of transient global ischemia (TGI), combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn2+ accumulation, and used transmission electron microscopy (TEM) to examine subcellular loci of the Zn2+ accumulation. In line with prior studies, TGI-induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4- than 1-hour recovery). Intriguingly, TEM examination of Timm's-stained sections revealed substantial Zn2+ accumulation in many postischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn2+ and the degree of their disruption were far greater at 4- than 1-hour recovery. These data provide the first direct characterization of Zn2+ accumulation in CA1 mitochondria after in vivo TGI, and support the idea that targeting these events could yield therapeutic benefits.

The neurotoxin beta-N-methylamino-l-alanine (BMAA) was first identified as a "toxin of interest" in regard to the amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC); studies in recent years highlighting widespread environmental sources of BMAA exposure and providing new clues to toxic mechanisms have suggested possible relevance to sporadic ALS as well. However, despite clear evidence of uptake into tissues and a range of toxic effects in cells and animals, an animal model in which BMAA induces a neurodegenerative picture resembling ALS is lacking, possibly in part reflecting limited understanding of critical factors pertaining to its absorption, biodistribution and metabolism. To bypass some of these issues and ensure delivery to a key site of disease pathology, we examined effects of prolonged (30day) intrathecal infusion in wild type (WT) rats, and rats harboring the familial ALS associated G93A SOD1 mutation, over an age range (80±2 to 110±2days) during which the G93A rats are developing disease pathology yet remain asymptomatic. The BMAA exposures induced changes that in many ways resemble those seen in the G93A rats, with degenerative changes in ventral horn motor neurons (MNs) with relatively little dorsal horn pathology, marked ventral horn astrogliosis and increased 3-nitrotyrosine labeling in and surrounding MNs, a loss of labeling for the astrocytic glutamate transporter, GLT-1, surrounding MNs, and mild accumulation and aggregation of TDP-43 in the cytosol of some injured and degenerating MNs. Thus, prolonged intrathecal infusion of BMAA can reproduce a picture in spinal cord incorporating many of the pathological hallmarks of diverse forms of human ALS, including substantial restriction of overt pathological changes to the ventral horn, consistent with the possibility that environmental BMAA exposure could be a risk factor and/or contributor to some human disease.

Valosin containing protein (VCP) mutations are the cause of hereditary inclusion body myopathy, Paget's disease of bone, frontotemporal dementia (IBMPFD). VCP gene mutations have also been linked to 2% of isolated familial amyotrophic lateral sclerosis (ALS). VCP is at the intersection of disrupted ubiquitin proteasome and autophagy pathways, mechanisms responsible for the intracellular protein degradation and abnormal pathology seen in muscle, brain and spinal cord. We have developed the homozygous knock-in VCP mouse (VCP^R155H/R155H) model carrying the common R155H mutations, which develops many clinical features typical of the VCP-associated human diseases. Homozygote VCP^R155H/R155H mice typically survive less than 21 days, exhibit weakness and myopathic changes on EMG. MicroCT imaging of the bones reveal non-symmetrical radiolucencies of the proximal tibiae and bone, highly suggestive of PDB. The VCP^R155H/R155H mice manifest prominent muscle, heart, brain and spinal cord pathology, including striking mitochondrial abnormalities, in addition to disrupted autophagy and ubiquitin pathologies. The VCP^R155H/R155H homozygous mouse thus represents an accelerated model of VCP disease and can be utilized to elucidate the intricate molecular mechanisms involved in the pathogenesis of VCP-associated neurodegenerative diseases and for the development of novel therapeutic strategies.

Introduction

Mutations in the valosin-containing protein (VCP) gene cause hereditary inclusion body myopathy (IBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently, these mutations have been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. A knock-in mouse model offers the opportunity to study VCP-associated pathogenesis.

Methods

The VCP(R155H/+) knock-in mouse model was assessed for muscle strength and immunohistochemical, Western blot, apoptosis, autophagy, and microPET/CT imaging analyses.

Results

VCP(R155H/+) mice developed significant progressive muscle weakness, and the quadriceps and brain developed progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies, and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons.

Conclusions

VCP(R155H/+) knock-in mice represent an excellent preclinical model for understanding VCP-associated disease mechanisms and future treatments.

Background

Pathogenic gain of function variants in Valosin-containing protein (VCP) cause a unique disease characterized by inclusion body myopathy with early-onset Paget disease of bone and frontotemporal dementia (also known as Multisystem proteinopathy (MSP)). Previous studies in drosophila models of VCP disease indicate treatment with VCP inhibitors mitigates disease pathology. Earlier-generation VCP inhibitors display off-target effects and relatively low therapeutic potency. New generation of VCP inhibitors needs to be evaluated in a mouse model of VCP disease. In this study, we tested the safety and efficacy of a novel and potent VCP inhibitor, CB-5083 using VCP patient-derived myoblast cells and an animal model of VCP disease.

Methods

First, we analyzed the effect of CB-5083 in patient-derived myoblasts on the typical disease autophagy and TDP-43 profile by Western blot. Next, we determined the maximum tolerated dosage of CB-5083 in mice and treated the 2-month-old VCP^R155H/R155H mice for 5 months with 15 mg/kg CB-5083. We analyzed motor function monthly by Rotarod; and we assessed the end-point blood toxicology, and the muscle and brain pathology, including autophagy and TDP-43 profile, using Western blot and immunohistochemistry. We also treated 12-month-old VCP^R155H/+ mice for 6 months and performed similar analysis. Finally, we assessed the potential side effects of CB-5083 on retinal function, using electroretinography in chronically treated VCP^R155H/155H mice.

Results

In vitro analyses using patient-derived myoblasts confirmed that CB-5083 can modulate expression of the proteins in the autophagy pathways. We found that chronic CB-5083 treatment is well tolerated in the homozygous mice harboring patient-specific VCP variant, R155H, and can ameliorate the muscle pathology characteristic of the disease. VCP-associated pathology biomarkers, such as elevated TDP-43 and p62 levels, were significantly reduced. Finally, to address the potential adverse effect of CB-5083 on visual function observed in a previous oncology clinical trial, we analyzed retinal function in mice treated with moderate doses of CB-5083 for 5 months and documented the absence of permanent ocular toxicity.

Conclusions

Altogether, these findings suggest that long-term use of CB-5083 by moderate doses is safe and can improve VCP disease-associated muscle pathology. Our results provide translationally relevant evidence that VCP inhibitors could be beneficial in the treatment of VCP disease.