Neurobiol Dis. 2016 Feb;86:29-40.

The carbonic anhydrase inhibitor methazolamide prevents amyloid beta-induced mitochondrial dysfunction and caspase activation protecting neuronal and glial cells in vitro and in the mouse brain.

Fossati S1, Giannoni P2, Solesio ME2, Cocklin SL2, Cabrera E2, Ghiso J3, Rostagno A4.
  • 1Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, United States. Electronic address:
  • 2Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, United States.
  • 3Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, United States; Department of Psychiatry, New York University School of Medicine, 550 First Avenue, New York, NY 10016, United States.
  • 4Department of Pathology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, United States. Electronic address:



Mitochondrial dysfunction has been recognized as an early event in Alzheimer’s disease (AD) pathology, preceding and inducing neurodegeneration and memory loss. The presence of cytochrome c (CytC) released from the mitochondria into the cytoplasm is often detected after acute or chronic neurodegenerative insults, including AD. The carbonic anhydrase inhibitor (CAI) methazolamide (MTZ) was identified among a library of drugs as an inhibitor of CytC release and proved to be neuroprotective in Huntington’s disease and stroke models. Here, using neuronal and glial cell cultures, in addition to an acute model of amyloid beta (Aβ) toxicity, which replicates by intra-hippocampal injection the consequences of interstitial and cellular accumulation of Aβ, we analyzed the effects of MTZ on neuronal and glial degeneration induced by the Alzheimer’s amyloid. MTZ prevented DNA fragmentation, CytC release and activation of caspase 9 and caspase 3 induced by Aβ in neuronal and glial cells in culture through the inhibition of mitochondrial hydrogen peroxide production. Moreover, intraperitoneal administration of MTZ prevented neurodegeneration induced by intra-hippocampal Aβ injection in the mouse brain and was effective at reducing caspase 3 activation in neurons and microglia in the area surrounding the injection site. Our results, delineating the molecular mechanism of action of MTZ against Aβ-mediated mitochondrial dysfunction and caspase activation, and demonstrating its efficiency in a model of acute amyloid-mediated toxicity, provide the first combined in vitro and in vivo evidence supporting the potential of a new therapy employing FDA-approved CAIs in AD.

KEYWORDS: Alzheimer’s disease; Amyloid; Carbonic anhydrase inhibitor; Caspase activation; Hippocampus; Hydrogen peroxide; Methazolamide; Microglia; Mitochondria; Neuron

PMID: 26581638



Mitochondrial dysfunction and the activation of caspases, proteases involved in the execution of programmed cell death, are increasingly recognized as important events in the pathology of Alzheimer’s disease (AD). Mitochondrial damage seems in fact to be one of the earliest events in the development of the disease.

Accordingly, recent work of our lab and others has demonstrated that intermediate aggregation species of Amyloid β (Aβ), the main component of parenchymal and vascular deposits in AD, engaged receptors on the cell membrane, called Death Receptors, triggering a chain of intracellular events leading to mitochondrial dysfunction, followed by activation of caspases and apoptotic cell death, in cerebral endothelial and neuronal cells in culture [1-4]. Therefore, inhibiting the release of pro-apoptotic molecules such as Cytochrome C (CytC) and Reactive Oxygen Species (ROS) from the mitochondria could result in prevention of cell death and amelioration of AD pathology in AD models and eventually in AD patients.

Methazolamide is a FDA approved carbonic anhydrase inhibitor (CAI) used in humans for many years for treatment of glaucoma, and can easily cross the blood brain barrier. This and analog drugs are also clinically used for the prevention of acute mountain sickness and related high altitude cerebral edema, confirming their efficacy in the brain and the safety of their systemic administration. In our previous studies on vascular and neuronal amyloid toxicity, a striking correlation was found between Methazolamide (MTZ) treatment, the preservation of mitochondrial membrane potential [4], and the reduction in CytC release from these organelles. Findings in animal models have also shown that caspase activation triggers early synaptic dysfunction in AD mouse models, suggesting that the preservation of healthy mitochondria is a key factor not only to prevent energetic failure in the AD brain, but also to inhibit the activation of caspases, such as caspase 9 and 3, thereby preventing synaptic dysfunction and the resulting memory loss. Hence, the possibility of blocking early mitochondrial damage in AD using MTZ, reducing activation of caspases and cell death, could represent an interesting approach in both prevention and therapy for AD.




Figure 1. Graphic representation of the prevention of Aβ-mediated mitochondrial dysfunction by MTZ. Aβ induces mitochondrial damage through activation of death receptors on the cell membrane (as we showed in [3]). Dysfunctional mitochondria release H2O2 and CytC, which contribute to the activation of Caspase 3 and to cell death. MTZ inhibits the release of H2O2, CytC and, as a consequence, the activation of Caspase 3.


Importantly, our study represents the first evidence of a protective effect of a MTZ against Aβ challenge in the mouse brain.

Drugs of the family that includes MTZ have a role in a number of physiological processes such as diuresis, production of body fluids, gluconeogenesis and lipogenesis. MTZ has also a high activity against mitochondrial CAs.

Our manuscript shows for the first time that MTZ is effective at inhibiting the overproduction of the mitochondrial ROS hydrogen peroxide (H2O2) induced by Aβ, suggesting that the ability of MTZ to reduce H2O2 production and prevent the associated mitochondrial CytC release could be the key mechanisms contributing to the prevention of caspase activation and the resulting apoptosis in our model.

Importantly, we showed that MTZ can effectively inhibit H2O2 production, CytC release and caspase activation not only in neuronal, but also in glial cells, decreasing the levels of active-caspase 3 in active microglia in the Aβ-injected mouse brain. Although mediated by different receptors, neuronal death and microglial activation drive spreading neurotoxicity. High levels of intracellular ROS might result in microglial over-activation and death. Indeed, recent reports demonstrated that oligomeric Aβ induces microglial neurotoxicity and IL-1β processing via production of mitochondrial ROS. Increased expression of active caspase-3 was also found in microglia of postmortem AD brain. An ideal therapeutic approach would involve attenuation of the microglial response to levels that are no longer deleterious, rather than the full elimination of the microglial response. This seems to be the case in our model in presence of MTZ, which preserves microglial activation while reducing the activation of caspase 3 below the threshold able to cause DNA fragmentation and cell death.

Thus, this treatment would have the potential to prevent neurodegeneration both directly, restoring mitochondrial function, and indirectly, avoiding excessive microglial activation and microglial cell death. Treatment studies using this compound on transgenic mouse models of AD are currently being performed in Dr. Fossati’s laboratory.

Overall, these findings support the potential of repurposing the FDA-approved drug MTZ as new therapeutic strategy for AD, emphasizing the importance of additional studies in this direction.




Figure 2. Neurotoxic signals induced by intra-hippocampal Aβ injection are inhibited by MTZ. Both neuronal and glial caspase 3 was activated in the hippocampus after Aβ injection. Intraperitoneal injection of MTZ 1 hour before Aβ prevented caspase activation in both cell types, and inhibited the resulting neurodegeneration.



  1. Fossati, S., et al., Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-beta variants in cells composing the cerebral vessel walls. FASEB J, 2010. 24(1): p. 229-41.
  2. Fossati, S., J. Ghiso, and A. Rostagno, Insights into Caspase-Mediated Apoptotic Pathways Induced by Amyloid-beta in Cerebral Microvascular Endothelial Cells. Neurodegenerative Diseases, 2012. 10(1-4): p. 324-328.
  3. Fossati, S., J. Ghiso, and A. Rostagno, TRAIL death receptors DR4 and DR5 mediate cerebral microvascular endothelial cell apoptosis induced by oligomeric Alzheimer’s Abeta. Cell Death Dis, 2012. 3: p. e321.
  4. Fossati, S., et al., Differential contribution of isoaspartate post-translational modifications to the fibrillization and toxic properties of amyloid beta and the Asn23 Iowa mutation. Biochem J, 2013. 456(3): p. 347-60.



This work was supported by grants from the National Institutes of Health, the American Heart Association, the Alzheimer’s Association, and the Blas Frangione Foundation. We thank Ludovic Debure for the design of the images for this commentary.



Silvia Fossati, PhD

Assistant Professor (Research) of Neurology and Psychiatry

Leon Levy Neuroscience Fellow

Director of the Biofluid Biomarker Core of the NYU Cohen Veteran Center

NYU School of Medicine

New York, NY 10016

Tel: 646 754 7327





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