CONCLUSIONES Y RECOMENDACIONES

As with the other techniques described in Section 1.1, the goal for CEST imaging is to identify “bioresponsive” agents that provide non-invasive measures of biochemical processes in vivo. There are exogenous agents that have been developed for CEST

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imaging, such as paramagnetic CEST (PARACEST) agents that create large Δω, permitting selective saturation of molecules with fast exchange rates (Zhang, Merritt et al. 2003, Woessner, Zhang et al. 2005, Zhang, Malloy et al. 2005, Woods, Donald et al. 2006). PARACEST agents provide information on processes like enzymatic activity or redox state changes, which change the properties of exchangeable protons and, by extension, the CEST signal (Zhang, Merritt et al. 2003, Aime, Carrera et al. 2005, Woods, Donald et al. 2006, Yoo and Pagel 2006, Pikkemaat, Wegh et al. 2007, Yoo, Raam et al. 2007, Chauvin, Durand et al. 2008, Viswanathan, Ratnakar et al. 2009). Although these exogenous agents are useful, like the other contrast agents described in the introduction, they must be injected into the subject for in vivo imaging. This is undesirable for many reasons, including invasiveness, cost, and concern with possible organ toxicity.

The goal for this dissertation was to further identify and develop endogenous CEST agents, primarily to avoid the complications that arise when injecting exogenous agents. To date, several types of endogenous metabolites have been imaged using CEST. In order to image an endogenous metabolite with CEST, it must exist at a high concentration (mM) in addition to having the optimal exchange conditions and chemical shift as described above. It must also contain exchangeable protons. Typically, these are amide (- NH), amine (-NH2), or hydroxyl (-OH) protons (Zhou, Blakeley et al. 2008, Haris, Cai et al. 2011, Jia, Abaza et al. 2011, Singh, Haris et al. 2012, Klomp, Dula et al. 2013, Yadav, Xu et al. 2014). These protons on various metabolites have been imaged with CEST to study cancer, neurological disorders, and muscle energetics.

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Amide CEST, or amide proton transfer (APT) imaging has proven useful in imaging of tumors that contain a high concentration of mobile macromolecular protons (Zhou, Lal et al. 2003). APT imaging provides a means of measuring changes in concentration of cytosolic proteins due to the CEST contrast from the slow-exchanging amide protons of peptide backbones. It also reports on the tissue pH, which is decreased in some tumor types and in ischemic tissue. The chemical exchange rate is highly dependent on tissue pH and so a change in the APT signal is observed when the pH is decreased (Zhou, Payen et al. 2003, Zhou, Blakeley et al. 2008, Jia, Abaza et al. 2011). However, when both pH and mobile macromolecular proton content changes, it is difficult to assess the individual contributions to APT.

In our effort to further develop CEST for study of cellular metabolism, we have chosen to focus on small metabolites. Metabolite levels are involved in cellular regulation of metabolism (Zimmerman 2005). Although overall cellular metabolic pathways are complex, they share common metabolites that are highly regulated to maintain proper cellular function (Metallo and Vander Heiden 2013). In turn, local concentrations of metabolites provide short-term regulation of enzymatic activity (Wegner, Meiser et al. 2015). The goal of our utilization of the CEST technique is to image metabolites that are altered in a way that is reflective of disruptions in metabolism, thus providing another tool for probing metabolic diseases.

There have been several recent publications on imaging of small metabolites from the Reddy lab (Haris, Cai et al. 2011, Haris, Singh et al. 2013). Amine protons of glutamate have been imaged with CEST (Cai, Haris et al. 2012, Kogan, Singh et al. 2013) (Figure

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1.3) and validated by NMR spectroscopy. With glutamate CEST (GluCEST), the variation in glutamate between gray and white matter in healthy human brain is clearly visible.

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Figure 1.3 GluCEST imaging and 1_{H MRS from a healthy human brain acquired at}

7T. A) anatomic proton image of an axial slice in human brain, showing the two voxel locations where spectroscopic data was acquired separately in the gray and white matter; B) B1 and B0 corrected GluCEST contrast map; C) map of distribution volumes (DVs) of

metabotropic Glu receptor subtype 5 from a PET image (Reprinted by permission of the Society of Nuclear Medicine: J Nucl Med31, Figure 2, copyright (2007)). The GluCEST map and the PET image show similar distribution pattern of Glu in brain, which is higher in GM compared to WM; D) B0 map and E) B1 map corresponding to the slice in A), and

used to correct the final CEST map for field inhomogeneity; F) 1H MRS spectra obtained from the voxels of gray matter and white matter in A). These spectra show higher amplitude of Glu –CH2 resonance (at 2.3 ppm.) and –CH resonance (at 3.75 ppm) in gray

matter; G) saturation pulse duration dependence of GluCEST data from human brain. H,I) z-spectra and corresponding asymmetry plots from human gray matter and white matter regions. The GluCEST peak at 3 ppm is ~11% from gray matter and ~7% from white matter. (Reprinted with permission (Cai, Haris et al. 2012).)

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GluCEST has also been used to study brain neurotransmitter alterations in an Alzheimer’s disease (Alz) mouse model (Figure 1.4) (Haris, Nath et al. 2013, Crescenzi, DeBrosse et al. 2014) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-based model of Parkinson’s (Bagga, Crescenzi et al. 2016). We also implemented the GluCEST technique in a longitudinal study, demonstrating the ability of the technique for measuring changes in GluCEST within a cohort of Alz mice over time (Crescenzi, DeBrosse et al. 2017). These studies have shown progressive loss of glutamate associated with neurodegenerative disease due to metabolic dysfunction. This has also provided some exciting evidence to support the concept of glutamate excitotoxicity in neurodegenerative diseases (Bagga, Crescenzi et al. 2016). Glutamate CEST (GluCEST) has also been used to study neuropsychiatric disorders such as epilepsy (Davis, Nanga et al. 2015) and schizophrenia (Roalf, Nanga et al. 2017). Overall, GluCEST has promise as a biomarker for metabolic changes, especially in neuropathologies.

Figure 1.4 4 GluCEST maps and corresponding immunohistochemistry (IHC). A–C

i) anatomical, coronal-slice images in a mouse brain corresponding to the GluCEST slice through the mid-hippocampus. A–C ii) GluCEST maps in a wild-type and in a progressive tauopathy model of Alzheimer’s (PS19) mice reveal lower glutamate levels throughout the brain of tauopathy mice, which continues to decrease with disease

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severity. A–C iii) The corresponding immunohistochemistry in the same slices of post- mortem brain tissue stained for hyper-phosphorylated tau (AT8) reveal varying severity of pathological tau protein within the PS19 cohort and show progressive loss of synapses. (Neuron staining (NeuN) and synapse staining (Syn) were both performed on the ex vivo tissue.) This is a clear example of the utility of the CEST method: imaging correlated to a standard technique provides more detailed insight into the biochemical pathways during the progression of disease.

In this work, we focused on CEST imaging of the amine protons of creatine to study oxidative phosphorylation (OXPHOS) and the hydroxyl protons of lactate to study glycolysis, which will be the subject of the following chapters. As discussed above, compared to classic spectroscopic methods for measuring metabolites in vivo, CEST has much higher spatial resolution (van Zijl and Yadav 2011). This is particularly beneficial for studying metabolic disruption, for example, in tumors that have heterogeneous tissue, or in skeletal muscle where specific muscle fiber types may be affected.