Differently increased volumes of multiple brain areas in Npc1 mutant mice following various drug treatments

Frontiers in Neuroanatomy 01 frontiersin.org Differently increased volumes of multiple brain areas in Npc1 mutant mice following various drug treatments VeronicaAntipova 1,2, DianaHeimes 1,3, KatharinaSeidel 1,4, JenniferSchulz 1, OliverSchmitt 1,5, CarstenHolzmann 6,7, ArndtRolfs 8, Hans-JürgenBidmon 9,10, EstibalizGonzálezdeSanRománMartín 10,11, PitterF.Huesgen 10,12, KatrinAmunts 9,13, JonasKeiler 1, NielsHammer 2,14,15, MartinWitt 1,16,17 and AndreasWree 1,7* 1 Institute of Anatomy, Rostock University Medical Center, Rostock, Germany, 2 Division of Macroscopic and Clinical Anatomy, Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Medical University of Graz, Graz, Austria, 3 Department of Oral and Maxillofacial Surgery, University Medical Center Mainz, Mainz, Germany, 4 Klinik für Frauenheilkunde und Geburtshilfe, Dietrich-Bonhoeffer-Klinikum, Neubrandenburg, Germany, 5 Department of Anatomy, Medical School Hamburg, University of Applied Sciences and Medical University, Hamburg, Germany, 6 Institute of Medical Genetics, Rostock University Medical Center, Rostock, Germany, 7 Centre of Transdisciplinary Neuroscience Rostock, Rostock, Germany, 8 Medical Faculty, University of Rostock, Rostock, Germany, 9 Institute of Neurosciences and Medicine, Structural and Functional Organisation of the Brain (INM-1), Forschungszentrum Jülich, Jülich, Germany, 10 Central Institute of Engineering, Electronics and Analytics, ZEA-3, Forschungszentrum Jülich, Jülich, Germany, 11 Centro Joxe Mari Korta, Donostia, Spain, 12 Institut für Biologie II, AG Funktional Proteomics, Freiburg, Germany, 13 C. and O. Vogt Institute for Brain Research, University Hospital Düsseldorf, University Düsseldorf, Düsseldorf, Germany, 14 Department of Orthopedic and Trauma Surgery, University of Leipzig, Leipzig, Germany, 15 Division of Biomechatronics, Fraunhofer Institute for Machine Tools and Forming Technology, Dresden, Germany, 16 Department of Anatomy, Technische Universität Dresden, Dresden, Germany, 17 Department of Anatomy, Institute of Biostructural Basics of Medical Sciences, Poznan Medical University, Poznan, Poland Background: Niemann-Pick disease type C1 (NPC1, MIM 257220) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. A murine model of Npc1−/− displays a rapidly progressing form of Npc1 disease, which is characterized by weight loss, ataxia, and increased cholesterol storage. Npc1−/− mice receiving a combined therapy (COMBI) of miglustat (MIGLU), the neurosteroid allopregnanolone (ALLO) and the cyclic oligosaccharide 2-hydroxypropyl-βcyclodextrin (HPßCD) showed prevention of Purkinje cell loss, improved motor function and reduced intracellular lipid storage. Although therapy of Npc1−/− mice with COMBI, MIGLU or HPßCD resulted in the prevention of body weight loss, reduced total brain weight was not positively influenced. Methods: In order to evaluate alterations of different brain areas caused by pharmacotherapy, fresh volumes (volumes calculated from the volumes determined from paraffin embedded brain slices) of various brain structures in shamand drug-treated wild type and mutant mice were measured using stereological methods. Results: In the wild type mice, the volumes of investigated brain areas were not significantly altered by either therapy. Compared with the respective wild types, fresh volumes of specific brain areas, which were significantly reduced in sham-treated Npc1−/− mice, partly increased after the pharmacotherapies in all OPEN ACCESS EDITED BY Jon I. Arellano, Yale University, UnitedStates REVIEWED BY Jason Newton, Virginia Commonwealth University, UnitedStates Maria Teresa Fiorenza, Sapienza University of Rome, Italy *CORRESPONDENCE Andreas Wree andreas.wr[email protected] RECEIVED 10 May 2024 ACCEPTED 01 July 2024 PUBLISHED 16 July 2024 CITATION Antipova V, Heimes D, Seidel K, Schulz J, Schmitt O, Holzmann C, Rolfs A, Bidmon H-J, González de San Román Martín E, Huesgen PF, Amunts K, Keiler J, Hammer N, Witt M and Wree A (2024) Differently increased volumes of multiple brain areas in Npc1 mutant mice following various drug treatments. Front. Neuroanat. 18:1430790. doi: 10.3389/fnana.2024.1430790 COPYRIGHT © 2024 Antipova, Heimes, Seidel, Schulz, Schmitt, Holzmann, Rolfs, Bidmon, González de San Román Martín, Huesgen, Amunts, Keiler, Hammer, Witt and Wree. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. TYPE Original Research PUBLISHED 16 July 2024 DOI 10.3389/fnana.2024.1430790 Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 02 frontiersin.org treatment strategies; most pronounced differences were found in the CA1 area of the hippocampus and in olfactory structures. Discussion: Volumes of brain areas of Npc1−/− mice were not specifically changed in terms of functionality after administering COMBI, MIGLU, or HPßCD. Measurements of fresh volumes of brain areas in Npc1−/− mice could monitor region-specific changes and response to drug treatment that correlated, in part, with behavioral improvements in this mouse model. KEYWORDS NPC1, mouse, lipid storage disorder, treatment, miglustat, 2-hydroxypropyl-βcyclodextrin, fresh volumes, brain areas 1 Introduction Niemann–Pick type C (NPC) (NPC1, MIM 257220) disease is a rare, genetically determined, autosomal, recessive neurodegenerative, neurovisceral storage disorder caused by mutations in the NPC1 gene (95%) or, only rarely, in the NPC2 gene (5%) that lead to the progressive neurodegeneration of the central nervous system (Sleat etal., 2004; Millat etal., 2005; Vanier, 2010; Newton etal., 2018; Lee etal., 2020). The loss of function of the NPC1 protein results in the impairment of the regulation of cholesterol efflux and the dysfunction of cholesterol homeostasis. The Npc1-mediated dysfunction of lipid transport has severe consequences for all brain cells. Besides the cellautonomous contribution of neuronal Npc1, aberrant Npc1 signaling in brain cells is critical for the pathology (Malara etal., 2024). The lipid accumulation in the lysosomes and late endosomes is probably the crucial event in the disease pathogenesis, although the underlying mechanisms are not fully understood (Bi and Liao, 2010; Lloyd-Evans and Platt, 2010; Peake et al., 2011; Wheeler and Sillence, 2020; Burbulla etal., 2021). Interestingly, Nguyen etal. (2024) identified that phosphorylation of T286 on CaMKIIα and S1303 on NR2B increased in mutant animals. These phosphosites are said to be crucial to learning and memory and can trigger neuronal death by altering protein–protein interactions. In addition to cholesterol, the Npc1 lesion has also been shown to affect the metabolism of sphingolipids (Liscum and Klansek, 1998; Vanier and Suzuki, 1998; Vanier, 1999), resulting in intracellular accumulation of unesterified cholesterol and other composites in various tissues, including an accumulation of the gangliosides and sphingolipids in the brain (Siegel and Walkley, 1994; Vanier, 1999; Zervas etal., 2001; Garver etal., 2007). The metabolic changes are accompanied by gliosis and extensive loss of Purkinje cells in the cerebellum and degeneration of other central nervous compartments (Elleder etal., 1985; Pacheco and Lieberman, 2008; Tang etal., 2010; Patterson etal., 2012; Piroth etal., 2017). The clinical manifestations of NPC1 disease vary and often correlate with the age of onset, which can occur from the prenatal period well into adulthood (Wraith etal., 2009; Vanier, 2010; Patterson etal., 2012; Mengel etal., 2017; Patterson etal., 2017; Baxter etal., 2022). The most typical neuropsychiatric symptoms are: cerebellar ataxia, dysarthria, dysphagia, progressive dementia, cataplexy, seizures and dystonia, psychosis, paranoid delusions or schizophrenia and saccadic eye movement abnormalities or vertical supranuclear gaze palsy (Garver etal., 2007; Sévin etal., 2007; Vanier, 2010; Patterson etal., 2012; Mengel etal., 2017; Bonnot etal., 2019; Rego etal., 2019). Neuropathological changes in the limbic system, the entorhinal area, or the piriform cortex in NPC1 are largely unexplored; only a few studies examined the limbic system and, in particular, the hippocampus in NPC1-diseased patients. Walterfang etal. (2010) studied gray matter volume and white matter structural differences in 6 adult patients with NPC and 18 sexand age-matched controls. The NPC patients showed bilateral gray matter reductions in large clusters in bilateral hippocampus, thalamus, upper cerebellum and insula, in addition to smaller regions of inferior-posterior cortex. Moreover, patients showed widespread reductions in fractional anisotropy in main pathways of white matter. Subsequent analysis suggests that these changes are caused by both impaired myelination and altered axonal structure. In a more recent study, the same group (Walterfang etal., 2013) compared 10 adult patients with NPC disease (18–49 years of age) with 27 ageand sex-matched controls. Most structures were smaller in patients with NPC (NPC1 and NPC2 not differentiated) compared with controls. The thalamus, hippocampus, and striatum showed the greatest and most significant volume reductions, and the left hippocampal volume correlated with symptom score and cognition. Vertex analysis of the thalamus, hippocampus and caudate implicated regions involved in memory, executive function, and motor control. The results from vertex analyses also showed a significant decrease in volume of the CA1 region and the subiculum (Walterfang et al., 2013). CA3, on the other hand, showed no morphological changes. The nucleus basalis and the septum showed severely atrophied pyramidal cell terminals. The cell bodies were almost completely absent (Ong etal., 2001). Other neuropathological studies in NPC1 patients also showed neurofibrillary tangles in the basal ganglia, brainstem, thalamus, and hippocampus, similar to those found in Alzheimer’s dementia (Love etal., 1995; Suzuki etal., 1997). So far, there is no causal therapy for NPC1, and the treatment efforts are focused on slowing the disease progression in man and/or mouse (Bräuer etal., 2019; Cariati etal., 2021; Rodriguez-Gil etal., 2021; Baxter etal., 2022; Campbell etal., 2023). Therapy with the iminosugar glucosylceramide synthase inhibitor N-butyldeoxynojirimycin (miglustat, Zavesca®) correlates with reduced glycosphingolipid levels, stabilized neurological phenotypes, slowing of disease progression and significantly reduced risk of mortality in NPC1 (Platt and Jeyakumar, 2008; Ginocchio etal., 2013; Fecarotta etal., 2015; Patterson etal., 2020; Curelaru etal., 2021). Miglustat (MIGLU) is the only treatment approved for NPC in Europe, Canada, and Japan (Lachmann and Platt, 2001; Lachmann, 2006; Patterson etal., 2007; Platt and Jeyakumar, 2008; Wraith etal., 2009). A positive effect of Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 03 frontiersin.org cyclodextrin (2-hydroxypropyl-β-cyclodextrin, HPβCD), a cyclic oligosaccharide believed to help transport cholesterol out of the lysosome (Rosenbaum etal., 2010; Taylor etal., 2012), has also been confirmed by clinical studies in NPC1 patients after intrathecal administration (Liu etal., 2010; Ramirez etal., 2010; Matsuo etal., 2013). In Npc1 −/− mice, a promising therapy is a combination of MIGLU, HPβCD, and the neurosteroid allopregnanolone (ALLO), leading to a prevention of cerebellar Purkinje cell loss, a significantly improved motor function, a reduced intracellular lipid storage, and an increased lifespan (Zervas etal., 2001; Ahmad etal., 2005; Davidson etal., 2009; Hovakimyan etal., 2013a; Maass etal., 2015; Tanaka etal., 2015; Davidson etal., 2016; Meyer etal., 2017; Ebner etal., 2018; Bräuer etal., 2019). In a mouse model, werecently compared the therapeutic effects of the combination therapy (COMBI) with that of MIGLU alone, HPßCD alone, and Sham injections on body and brain weight and the behavior of Npc1−/− mice in a larger cohort (Holzmann etal., 2021). The results showed that each drug had the potential to significantly improve body weight deficits in Npc1−/− mice; however, treatments with COMBI, MIGLU and HPßCD did not significantly increased brain weight in Npc1−/− mice, their values being increased by 3.58, 0.51 and 7.67%, respectively, compared with Shamtreated mutants (Holzmann etal., 2021). Here, weused the BALB/cNctr-Npc1m1N/−J Jackson Npc1 mouse strain, which carries a spontaneous mutation of Npc1 and partly displays pathological hallmarks of the human disease (Higashi etal., 1993; Pentchev, 2004; Maass etal., 2015), resulting in weight loss, increased lipid storage, ataxia, and progressive neurodegeneration characterized by cerebral and cerebellar atrophy, hypomyelination, degeneration of neurons in various parts of the brain, the most pronounced being cerebellar Purkinje cells (Morris etal., 1982; Tanaka etal., 1988; Higashi etal., 1993; Zervas etal., 2001; Loftus etal., 2002; Võikar etal., 2002; Sarna etal., 2003; Walkley and Suzuki, 2004; Luan et al., 2008). The cerebral cortex of Npc1 −/− mice showed mild to moderate signs of degeneration seen in the prefrontal cortex by a significant neuron reduction of 28% and volume reduction of 19% (German etal., 2001a,b). Loss of neurons was also observed in the somatosensory and motor cortices (German etal., 2001a,b; Ohara etal., 2004; Pressey etal., 2012), striatum, globus pallidus, medial geniculate body, cerebellar cortex besides the Purkinje neurons and deep cerebellar nuclei (Ong etal., 2001; Maass etal., 2015); these also led to volume reductions (Tanaka etal., 1988; German etal., 2001a,b). Other affected brain regions of Npc1 −/− mice were thalamus and brainstem with loss of neurons in substantia nigra, nucleus tractus solitarii, and locus coeruleus (German etal., 2002; Luan etal., 2008; Chiba etal., 2014). As far as was studied in various white matter regions of Npc1−/− mice, reduced volumes were explained by a degeneration of myelin sheaths (Lope-Piedrafita etal., 2008), supporting the assumption that Npc1 mutation causes hypomyelination (German et al., 2002). A significant reduction of myelination, especially in the olfactory bulb, cerebral cortex, hippocampus, thalamus, and hypothalamus, was reported by Qiao etal. (2018). Most obvious was hypomyelination by the reduction in height of the corpus callosum in cross sections of the Npc1 −/− mouse brain, accompanied by a reduction in the number of glial cells by 63% (German etal., 2001b, 2002; Ahmad etal., 2005). Although various quantitative data on the brains and parts thereof in untreated Npc1 −/− mice and respective wild types have been described in the last decades (Supplementary Table S1), a systematic evaluation of regionand areal-specific volumes for comparison with effects of drug treatments in Npc1 +/+ and Npc1 −/− mice is lacking. Therefore, in the present study, weanalyzed volumes of brain regions and areas of the BALB/cNctr-Npc1m1N/−J Jackson Npc1 mouse strain in Npc1+/+ and Npc1−/− mice that received either Sham or different drug therapies. Importantly, for the first time, Npc1−/− mice treated with (i) vehicle injection (Sham), (ii) a combination of MIGLU, ALLO, and HPßCD (COMBI), (iii) MIGLU monotherapy (MIGLU), and (iv) HPßCD monotherapy (HPßCD) were included in the evaluation in order to look for drug-related therapeutic effects. For comparison, four respective Npc1 +/+ control mouse groups were studied in parallel. Because no noticeable lateral differences were detected in random measurement, data were evaluated in the right hemispheres only. The most important point, however, is that all data presented are given as fresh volumes, i.e., all measures were corrected for individual brainspecific tissue shrinkage during the histological workup. Accordingly, the aim of the present work is to firstly evaluate which parts of the brain are affected by volume loss in Npc1 −/− mice compared with Npc1+/+ mice, and, secondly, whether volumes of different parts of the brain of Npc1 −/− mice are affected differently by the different therapies. To do so and to get an overview, brains were divided into 13 structural parts and their fresh volumes determined. In addition, due to olfactory (Hovakimyan etal., 2013b; Meyer etal., 2017, 2018; Witt etal., 2018; Bräuer etal., 2019) and learning (Võikar etal., 2002; Hovakimyan etal., 2013a; Bräuer etal., 2019; Holzmann etal., 2021) deficits in Npc1 −/− mice, functionally associated areas were examined more closely. 2 Results At first glance, the perfusion-fixed brains of Npc1 −/− mice appeared smaller than those of Npc1 +/+ mice. The olfactory bulb in particular appeared smaller and the cerebellar folia less prominent. Screening of the Nissl-stained specimens at low microscopic magnification already showed differences in brain cytoarchitecture between Npc1 −/− compared with Npc1 +/+ mice (Figures1A–F). The olfactory bulb of Npc1 −/− mice in toto was smaller, mainly due to the reduced white matter structures. Also, the cross-sectional area of the accessory olfactory bulb of Npc1−/− mice was reduced (Figure1B). When examining the preparations of the Npc1 −/− mice, the generally narrowed profiles of the subcortical white matter and partly reduced thickness of the cortical areas were particularly noticeable (Figures1C–F). Moreover, the density of glia cell nuclei in the white matter structures appeared to be lower (Figures1C–F). As already seen when dissecting the cerebellum out of the skull of Npc1 −/− mice, the respective histological slices showed a smaller cerebellum-containing area compared with Npc1+/+ mice. In Table1, the measurements of the analyzed brain structures found in Npc1+/+ and Npc1−/− mice of the Sham groups are summarized, the data given as fresh volumes (mm 3 ), or as maximal heights (mm) for the anterior commissure and the corpus callosum, as seen in frontal sections. 2.1 Shrinkage factor In order to obtain a reliable, quantitative fresh volume calculation from paraffin-embedded brain material, the volume of the sectioned brain was corrected for the brain-specific shrinkage (Zilles, 1978; Wree etal., 1981; Beck etal., 1993). The shrinkage factors of the brains of all individual 8 experimental groups did not differ significantly nor did brains of the overall groups of Npc1 +/+ versus Npc1 −/− mice showed Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 04 frontiersin.org significant differences (Two-way ANOVA, genotype p = 0.553, treatment p = 0.430; Figures2A,B). Testing the correlation between the individual brain weights and the respective individual shrinkage factors in 49 samples (Figure2D) according to Spearman revealed a correlation coefficient of −0.277, and a p-value of 0.0539. However, as there is a low correlation between brain weight and shrinkage factor, it seems justified to speculate about a relationship between both parameters. During embedding of the fixed brains in paraffin wax especially water and lipids were removed by the solvents used. It could behypothesized that the smaller brains of Npc1 −/− mice, which contain some lipids in lower amounts per gram of brain, but higher concentrations of various proteins, shrink less (Santiago-Mujica etal., 2019; Gläser etal., 2020). 2.2 Drug treatment partially altered fresh volumes of brain regions of Npc1+/+ and Npc1−/− mice 2.2.1 COMBI treatment In Npc1+/+ mice, COMBI treatment reduced fresh volumes of the frontal (−25.3%) (Figure3A), motor (−36.2%) (Figure3B) and sensory FIGURE1 Nissl-stained frontal sections of a Npc1+/+ mouse (A,C,E) at the levels with respect to Bregma: +3.08 (A), −0.10 (C), and −2.06 (E). The respective sections of the Npc1−/− mouse (B,D,F) were chosen as best corresponding to (A,C,E). (A,B) sections at the level of the largest extent of the accessory olfactory bulb, (C,D) sections at the level of the largest height of the anterior commissure, (E,F) sections at the level of the rostral beginning of the fasciola cinereum. Scale bar: 1 mm for (A–F). TABLE1 Mean values ± SEM of the fresh volumes (right hemisphere) and heights of Sham-treated Npc1+/+ and Npc1−/− mice, and relative changes of Npc1−/− mice compared with Npc1+/+ mice. Parameter Npc1+/+ Npc1−/−Rel. change Body weight (g) 24.826 ± 0.573 13.511 ± 0.633*** −45.6% Brain weight (g) 0.474 ± 0.006 0.374 ± 0.007*** −21.1% Brain volume (mm3)0.459 ± 0.006 0.362 ± 0.007 *** −21.1% Basal forebrain volume (mm3)62.819 ± 1.745 51.833 ± 1.645*** −17.5% CPu + GP + ic volume (mm3)19.910 ± 0.560 15.847 ± 0.528* −20.4% Mesencephalon volume (mm3)19.556 ± 0.814 14.974 ± 0.768*** −23.4% Brainstem volume (mm3)31.268 ± 0.965 28.044 ± 0.909* −10.3% Frontal cortex volume (mm3)16.923 ± 0.675 11.999 ± 0.636*** −29.1% Motor cortex volume (mm3)6.394 ± 0.318 3.678 ± 0.300*** −42.5% Sensory cortex volume (mm3)30.082 ± 0.995 21.686 ± 0.939*** −27.9% Insular cortex volume (mm3)5.487 ± 0.417 5.227 ± 0.394 −4.7% Hippocampus volume (mm3)23.463 ± 0.649 20.667 ± 0.612** −11.9% Cerebellum volume (mm3)29.196 ± 0.839 21.272 ± 0.791*** −27.1% Ventricle volume (mm3)1.892 ± 0.480 1.635 ± 0.452 −13.6% White matter volume (mm3)6.558 ± 0.239 3.190 ± 0.225*** −51.4% Anterior commissure volume (mm3)0.470 ± 0.033 0.178 ± 0.032*** −62.1% Corpus callosum height (μm) 418.7 ± 16.8 197.7 ± 17.7*** −52.8% Anterior commissure height (μm) 409.6 ± 21.4 309.9 ± 22.5** −24.3% Olfactory bulb volume (mm3)8.142 ± 0.219 5.335 ± 0.219*** −34.5% Accessory olfactory bulb vol. (mm3)0.461 ± 0.125 0.364 ± 0.125*** −21.0% Anterior olfactory nucleus vol. (mm3)0.727 ± 0.023 0.601 ± 0.023*** −17.3% Olfactory tubercle volume (mm3)1.890 ± 0.044 1.506 ± 0.046*** −20.3% Lateral olfactory tract volume (mm3)0.350 ± 0.012 0.208 ± 0.012*** −40.6% Piriform cortex volume (mm3)5.077 ± 0.140 3.992 ± 0.148*** −21.4% Medial habenular nucleus vol. (mm3)0.109 ± 0.003 0.099 ± 0.003*−9.1% Dentate gyrus volume (mm3)2.628 ± 0.076 2.184 ± 0.084*** −16.9% Cornu ammonis field 1 volume (mm3)3.962 ± 0.123 3.071 ± 0.136*** −22.5% Cornu ammonis field 2 + 3vol. (mm3)2.776 ± 0.086 2.536 ± 0.095 −8.6% Subiculum volume (mm3)2.222 ± 0.101 1.790 ± 0.111** −19.4% Pre- + parasubiculum volume (mm3)1.655 ± 0.089 1.459 ± 0.099 −11.8% Entorhinal cortex volume (mm3)4.863 ± 0.247 3.892 ± 0.274*−20.0% The double lines delineate the 13 regions into which the whole brains were divided. CPu + GP + ic = caudate putamen + globus pallidus + internal capsule. *Significant difference between Npc1+/+ and Npc1−/− mice. *p < 0.05, **p < 0.01, ***p < 0.001. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 05 frontiersin.org cortices (−17.8%) (Figure3C) compared with the Sham-treated group; the other regions, however, were left unaltered. In Npc1 −/− mice, COMBI induced in none of the 13 regions significantly changed fresh volumes (Figures4A–E, 3A–F) compared with Sham treatment. When both COMBI treatment groups were compared, significant differences of fresh volumes between Npc1 +/+ and Npc1 −/− mice were found in the brainstem (33.32 ± 1.11 mm 3 vs. 28.43 ± 1.11 mm 3 ), and cerebellum (27.74 ± 0.97 mm3 vs. 22.72 ± 0.97 mm3). 2.2.2 MIGLU treatment Compared with Sham treatment, MIGLU treatment of Npc1 +/+ mice did not induce significant changes of fresh volumes of any region investigated (Figures4A–E, 3A–F). In the respective Npc1 −/− mice, MIGLU treatment only resulted in a significant increase of fresh volumes in the CPu + GP + ic region (Sham: 15.85 ± 0.53, MIGLU: 19.04 ± 0.79) (Figure 4B), all other region remained unchanged (Figures4A,C–E, 3A–F). 2.2.3 HPßCD treatment Npc1 +/+ mice treated with HPßCD showed significantly reduced fresh volumes of the mesencephalon (−17.1%) (Figure4C), and frontal (−19.6%) (Figure 3A) and motor cortices (−36.9%) (Figure 3B) compared with Sham treatment. In Npc1 −/− mice, no significant changes of fresh volumes of any region compared with the respective Sham group were found (Figures4A–E, 3A–F). After HPßCD treatment, Npc1 −/− mice still showed significantly lower fresh volumes in the basal forebrain (51.91 ± 2.01 mm 3 vs. 62.83 ± 1.87 mm 3 ), CPu + GP + ic (17.03 ± 0.65 mm 3 vs. 20.52 ± 0.60mm3), brainstem (29.87 ± 1.11 mm3 vs. 34.23 ± 1.03 mm3), sensory cortex (24.40 ± 1.15 mm 3 vs. 28.72 ± 1.06 mm 3 ), hippocampus (21.86 ± 0.75 mm 3 vs. 24.21 ± 0.69 mm 3 ), and cerebellum (24.56 ± 0.97 mm 3 vs. 27.65 ± 0.90 mm 3 ) compared with the corresponding Npc1+/+ mice. 2.3 Fresh volumes of the white matter structures were reduced in Npc1−/− mice 2.3.1 COMBI treatment In Npc1+/+ mice, COMBI treatment reduced the fresh volume of the white matter (−19.9%) (Figure5A) compared with the Shamtreated group. In Npc1−/− mice, COMBI induced a significant increase in the white matter fresh volume (+ 42.8%) (Figure5A) compared with Sham treatment. When white matter and anterior commissure parameters of both COMBI treatment groups were compared, significant differences were found only in the height of the corpus callosum of Npc1 +/+ and Npc1 −/− mice (415.57 ± 21.77 μm vs. 264.95 ± 21.77 μm) (Figure5C). 2.3.2 MIGLU treatment Compared with Sham treatment, MIGLU treatment of neither Npc1 +/+ nor Npc1 −/− mice significantly changed white matter and anterior commissure parameters (Figures5A–D). 2.3.3 HPßCD treatment Npc1+/+ mice treated with HPßCD showed a significantly reduced fresh volume of the white matter (−20.5%) (Figure 5A), and corresponding reduced corpus callosum height (−34.8%, Figure5C), compared with Sham treatment. In Npc1 −/− mice we found no significant changes in white matter and anterior commissure parameters compared with the respective Sham group (Figures5A–D). After HPßCD treatment Npc1 −/− , mice still showed significantly lower fresh volumes of the white matter (4.01 ± 0.28 mm 3 vs. 5.21 ± 0.26 mm 3 ) and the anterior commissure (0.23 ± 0.04 mm 3 vs. 0.38 ± 0.04 mm 3 ) compared with the corresponding Npc1+/+ mice. 2.4 Fresh volumes of all olfactory areas were reduced in Npc1−/− mice compared with the respective sham-treated Npc1+/+ mice 2.4.1 Sham treatment In the Sham groups, all olfactory regions investigated showed significantly smaller fresh volumes in Npc1 −/− mice compared with the corresponding Npc1 +/+ ones: olfactory bulb (−34.5%) (Figure6A), accessory olfactory bulb (−21.0%) (Figure6B), anterior olfactory nucleus (−17.3%) (Figure 6C), olfactory tubercle (−20.3%) (Figure6D), lateral olfactory tract (−40.6%) (Figure6E), and piriform cortex (−21.4%) (Figure6F). 2.4.2 COMBI treatment COMBI treatment of Npc1 +/+ mice reduced fresh volumes of the accessory olfactory bulb (−25.4%) (Figure6B) and the olfactory tubercle (−16.4%) (Figure6D). All other olfactory subregions of Npc1 +/+ mice were left unaffected in these mice (Figures6A,C,D). In the COMBItreated Npc1 −/− mice, a significant increase of fresh volume compared with the Sham group was measured in the lateral olfactory tract only (Figures6A–F). When the COMBI treatment groups of Npc1 +/+ and Npc1 −/− mice were compared, significant differences of fresh volumes were only found in the olfactory bulb (7.29 ± 0.27 mm 3 vs. 5.95 ± 0.27 mm 3 ), and the lateral olfactory tract (0.349 mm 3 vs. 0.257 mm 3 ). 2.4.3 MIGLU treatment Compared with Sham treatment, MIGLU treatment of Npc1 +/+ mice resulted in a significant decrease of the fresh volume of the accessory olfactory bulb (Figure 6B). In Npc1 −/− mice, MIGLU treatment induced an increase of fresh volumes in the olfactory bulb, olfactory tubercle and the piriform cortex compared with the respective Sham group (Figures6A,D,F). Comparison of the MIGLU treatment groups of Npc1 +/+ and Npc1 −/− mice revealed a significant difference of fresh volumes in the olfactory bulb only (8.69 ± 0.66 mm3 vs. 6.41 ± 0.33 mm3). 2.4.4 HPßCD treatment HPßCD-treated Npc1+/+ mice showed significantly reduced fresh volume in the olfactory bulb (−11.6%) (Figure6B) and the accessory olfactory bulb (−11.7%) (Figure6A) compared with Sham treatment. In Npc1−/− mice, significant increases of fresh volumes were found in the olfactory bulb (+20.3%) (Figure6A), the lateral olfactory tract (+28.4%) (Figure6E), and piriform cortex (+20.5%) (Figure6F) compared with the respective Sham group. After HPßCD treatment, Npc1 +/+ mice still had significantly lower fresh volumes in the olfactory bulb (6.42 ± 0.27 mm 3 vs. 7.20 ± 0.25 mm 3 ), anterior olfactory nucleus (0.593 ± 0.028 mm 3 vs. 0.685 ± 0.026 mm 3 ), and lateral olfactory tract (0.267 ± 0.015 mm 3 vs. 0.338 ± 0.013 mm 3 ) compared with the corresponding Npc1+/+ mice. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 06 frontiersin.org 2.4.5 All treatments Irrespective of the drug applied, fresh volumes of the olfactory bulb and the lateral olfactory tract of Npc1 +/+ mice significantly exceeded those of the corresponding volumes of Npc1 −/− mice (Figures 6A,E). In all other structures, the corresponding fresh volumes of Npc1 +/+ mice did not differ significantly from the respective data of the Npc1−/− mice (Figures6B–D,F). 2.5 COMBI and MIGLU treatments partly increased fresh volumes of some hippocampal areas of Npc1−/− mice After Sham treatment, Npc1 +/+ mice had significantly larger fresh volumes than Npc1 −/− mice in 4 of the 6 limbic areas investigated: entorhinal cortex (4.86 ± 0.25 mm 3 vs. 3.89 ± 0.27 mm 3 ), dentate gyrus (2.63 ± 0.08 mm 3 vs. 2.18 ± 0.08 mm 3 ), cornu ammonis field 1 (3.96 ± 0.12 mm 3 vs. 3.07 ± 0.14 mm 3 ), and subiculum (2.22 ± 0.10 mm3 vs. 1.79 ± 0.11 mm3) (Figures7A–C,E). Respective measurements of cornu ammonis fields 2 + 3 (2.78 ± 0.09 mm 3 vs. 2.54 ± 0.10 mm 3 ) and presubiculum + parasubiculum (1.66 ± 0.09 mm 3 vs. 1.49 ± 0.10 mm 3 ) did not differ significantly (Figures7D,F). 2.5.1 COMBI treatment COMBI treatment of Npc1 +/+ mice had no significant effects on fresh volumes of the limbic areas (Figures7A–F). In the COMBItreated Npc1−/− mice, a significant increase of fresh volume compared with the Sham group was measured in the cornu ammonis field 1 by +28.5% and field 2 + 3 by +17.3% (Figures6C,D). When comparing FIGURE2 (A) Differentiated shrinkage factors of the brains of Npc1+/+ and Npc1−/− mice of the Sham, COMBI, MIGLU, and HPßCD groups, and (B) mean shrinkage factors of the brains of Npc1+/+ and Npc1−/− mice irrespective of treatment. (C) Brain weights of Npc1+/+ and Npc1−/− mice of the Sham, COMBI, MIGLU, and HPßCD groups, and (D) correlation analysis of brain weights and shrinkage factors of all brains investigated. * Significant post-hoc tests are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Data are means ± SEM. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 07 frontiersin.org COMBI treatment groups of Npc1+/+ and Npc1−/− mice, no significant differences in fresh volumes of hippocampal areas were detected (Figures7A–F). 2.5.2 MIGLU treatment Compared with Sham treatment, MIGLU treatment of Npc1 +/+ mice resulted in no significant changes of fresh volumes of either FIGURE3 Fresh volumes of (A) frontal cortex, (B) motor cortex, (C) sensory cortex, (D) insular cortex, (E) hippocampus, and (F) cerebellum of the brains of Npc1+/+ and Npc1−/− mice of the Sham, COMBI, MIGLU, and HPßCD groups. Significant post-hoc tests are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Data are means ± SEM. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 08 frontiersin.org limbic region (Figures7A–F). In respective Npc1−/− mice, significant MIGLU-based increases of fresh volumes compared with the Sham group was found in cornu ammonis field 1 (+ 28.3%) and subiculum (+ 46.1%) (Figures7C,E). When comparing MIGLU treatment groups of Npc1+/+ and Npc1−/− mice, there were no significant differences in the fresh volumes of the hippocampal areas (Figures7A–F). FIGURE4 Fresh volumes of (A) basal forebrain, (B) CPu + GP + ic, (C) mesencephalon, (D) brainstem, and (E) ventricle of the brains of Npc1+/+ and Npc1−/− mice of the Sham, COMBI, MIGLU, and HPßCD groups. Significant post-hoc tests are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Data are means ± SEM. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 09 frontiersin.org 2.5.3 HPßCD treatment Compared with Sham treatment, HPßCD treatment did not significantly change fresh volumes in either hippocampal region of Npc1 +/+ mice (Figures7A–F). The same held true for Npc1 −/− mice (Figures7A–F). Compared to the Sham-treated Npc1 −/− mice, after HPßCD treatment Npc1−/− mice did not show any further significantly lower fresh volumes in the entorhinal cortex, the dentate gyrus, the cornu ammonis field 1, and the subiculum (Figures7A–C,E). 2.6 The relative volumes of regions and areas of Npc1+/+ and Npc1−/− mice partly differed A comparison of relative volumes of regions and areas of Npc1+/+ and Npc1 −/− mice can shed light on whether structures are equally altered in both groups or whether part of the brain of Npc1−/− mice is significantly larger or smaller. 2.6.1 Sham treatment of Npc1+/+ and Npc1−/− mice The relative volumes of Sham-treated Npc1+/+ and Npc1−/− mice showed different patterns in different regions and areas. One pattern, seen in the CPu + GP + ic region and the mesencephalon, showed nearly identical relative volumes in both groups (Figures 8A,B). However, in the brainstem and hippocampus, significantly larger relative volumes were found in Npc1 −/− mice compared with Npc1 +/+ mice (Figures 8C,D). In contrast, significantly smaller relative volumes were observed in the motor cortex and cerebellum in Npc1 −/− mice (Figures8E,F). The most pronounced differences, however, were found in the neuron-free areas of the brain. In the Sham-treated groups, the relative volumes of the white matter were 2.58 ± 0.08% in Npc1 +/+ mice, and 1.59 ± 0.08% in Npc1 −/− mice (Figure 9A). In these groups, the anterior commissure showed an even more pronounced difference in relative volumes: in Npc1+/+ mice, the anterior commissure had a relative volume of 0.18 ± 0.01%, the respective Npc1 −/− mice 0.09 ± 0.01% (Figure9B). FIGURE5 Fresh volumes of (A) white matter and (B) anterior commissure; (C) maximal heights of corpus callosum, and (D) maximal heights of anterior commissure of the brains of Npc1+/+ and Npc1−/− mice of the Sham, COMBI, MIGLU, and HPßCD groups. Significant post-hoc tests are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). Data are means ± SEM. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 16 frontiersin.org amount of hypomyelination varied between different parts of the brain (Payne and Hales, 2004; Miller, 2013; Cole etal., 2019). Although the hypomyelination is evident, its contribution to the significant volume reductions in the various white and gray matter brain structures cannot berealistically assessed. Curiously, and as yet not explainable, there is no obvious myelin reduction in the optic tract (Figures12D,F). 3.3 Reduced fresh volumes represent one factor in functional disorders in Npc1−/− mice The spontaneous or forced behavior of a mouse is a complex interplay of various neurophysiological, molecular biological and neuroanatomical parameters, one of which is the fresh volume of the whole brain or specific brain structures. Many studies are being conducted to determine relationships between the volume of parts of the brain and behavior in humans and animals (Francx etal., 2016; Guma etal., 2017; Chen etal., 2024; Gong etal., 2024; Magalhaes etal., 2024). Here, weshowed increased fresh volumes of specific brain areas of Npc1−/− mice treated with various drugs that, at least in part, showed related improvements in behavioral tests. One should keep in mind, however, that the fresh volume of a brain area represents the sum of the number and size of neurons and glial cells and the amount of neuropil including the degree of myelination. 3.4 Motor and sensory cortices in Npc1+/+ and Npc1−/− mice Locomotor and complex motor behavior and the motor defects in Npc1−/− mice can beassigned to changes in motor and sensory areas. The Sham-treated mutant mice exhibited motor impairments in all performed tests, like open field, elevated plus maze and accelerod tests (Võikar etal., 2002; Hovakimyan etal., 2013a; Hung etal., 2016; Santiago-Mujica etal., 2019; Holzmann etal., 2021). The disproportionate reduction in fresh volumes in the motor and sensory areas as well as the reduced cerebellar volume including the Purkinje neuron loss (Maass etal., 2015) in Npc1−/− mice compared to Npc1 +/+ mice could bethe structural basis of these functional deficits. Given the greatly reduced interhemispheric projections passing through the small corpus callosum, inadequate motor coordination as seen in Npc1−/− mice could bealso expected. 3.4.1 Frontal cortices in Npc1+/+ and Npc1−/− mice The frontal cortex is essential for decision-making, goal-directed behaviors, and emotional and cognitive processes (Graham etal., 2021; Bukwich etal., 2023). Data measured in open field tests were designed to reflect the abilities of spontaneous locomotion (total distance, total visits) and anxiety (central/total ratio). Npc1 −/− mice were more anxious than Npc1 +/+ mice (Holzmann etal., 2021) and cognitive impairment of Npc1 −/− mice was noticed in water maze tests (Võikar etal., 2002; Hovakimyan etal., 2013a). The obviously worse outcomes of these tests in Npc1 −/− mice may bedue to reduced fresh volume and hypomyelination of respective brain areas (Figures3A, 11A). 3.4.2 Olfactory areas and olfaction in Npc1+/+ and Npc1−/− mice Impaired olfaction, known to occur in many neurodegenerative diseases, may also constitute a key symptom in Npc1. In previous studies, wefound that the peripheral branch of the olfactory system, namely the olfactory mucosa, was severely damaged (Hovakimyan etal., 2013b). Olfactory receptor neuron numbers were reduced, proliferating neuroepithelial progenitor cells increased (Meyer etal., 2017), and olfactory receptor expression reduced (Meyer etal., 2018). Wealso demonstrated massive myelin-like inclusions in olfactory ensheathing cells (olfactory glia) as well as astrogliosis in the glomerular layer of the olfactory bulb, the first central relay of the central olfactory system (Hovakimyan etal., 2013b). The reduced olfactory bulb volume (Figures6A,B) is difficult to evaluate, because the increased amount of GM3 (Figure10) can beoutweighed by decreased myelination as seen by the ST [ST (18:1/24:1)] (Figure10). Myelination in the olfactory bulb begins in the outer plexiform layer and is most pronounced in the lateral olfactory tract, which constitutes only a small central portion of the olfactory bulb. The FIGURE12 Myelin staining by the Gallyas method of frontal sections of an Npc1+/+ mouse (A,C,E) at the levels with respect to Bregma: +3.08 (A), −0.10 (C) and − 2.06 (E). The respective sections of the Npc1−/− mouse (B,D,F) were chosen as best corresponding to (A,C,E). (A,B) Section at the level of the largest extent of the accessory olfactory bulb, (C,D) section at the level of the largest height of the anterior commissure, (E,F) section at the level of the rostral beginning of the fasciola cinereum. The scale bar presents 1 mm for (A–F). Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 17 frontiersin.org most significant differences between groups occur in olfactoryrelated structures containing reasonable amounts of myelin (Figures 12A,B). Both central parts (olfactory bulb, accessory olfactory bulb, and lateral olfactory tract) of the central olfactory system have significantly reduced volumes, which may bedue, among other factors, to hypomyelination. Thus, this also may bea key factor in impaired olfactory acuity of Npc1 −/− mice, as evaluated in behavior tests (Meyer etal., 2018). 3.4.3 Therapeutic interventions affected brain regions and areas of Npc1+/+ and Npc1−/− mice differently Relative changes of fresh volumes and heights of brain regions and areas of Npc1 +/+ and Npc1 −/− in the Sham groups revealed a mean reduction of 24.38% in Npc1 −/− mice compared with the respective Npc1 +/+ mice (Figure11A). Looking at the various drugs, the most beneficial effect was seen following MIGLU treatment with a mean relative difference of 7.02% (Figures11B,D). Relative changes of fresh volumes and heights of brain regions and areas of Npc1 +/+ and Npc1 −/− mice of the COMBI, MIGLU, and HPßCD treatment groups compared to their respective Sham treatment group differed remarkably. Differences were noted between the same treatments administered to Npc1 +/+ and Npc1 −/− mice. Whereas COMBI treatment in Npc1 +/+ mice reduced these parameters by 3.72% (Figure13A), this treatment in Npc1−/− mice increased fresh volume and heights by 13.85% (Figure13B). More differentiated results were found in MIGLU treatment groups. Whereas MIGLU treatment in Npc1+/+ mice only minimally reduced these parameters by 2.63% (Figure 13C), MIGLU treatment in Npc1 −/− mice significantly increased fresh volume and heights by 20.04% (Figure13D). Compared with Sham-treated Npc1+/+ mice, HPßCD treatment reduced these parameters by 7.01% (Figure13E); however, HPßCD treatment in Npc1−/− mice significantly increased the mean value by 11.66% (Figure13F). In line with the present findings, a positive effect of single HPßCD injection at P7 Npc1 −/− mice was also seen by Nusca etal. (2014) at P28 as a partly rescue of normally occurring cerebellum size reduction. The same group reported a positive effect of HPßCD in Npc1 +/+ mice, injected at P4, P7, and thereafter weekly up to P49 and investigated at P75, a partly recue of myelinated fibers in the primary visual cortex and optic nerve compared with sham-treated Npc1 +/+ mice. Myelin rescue was accompanied by a normalization of cellularity and cytoarchitecture (Palladino etal., 2015). It is controversial whether HPBCD effectively crosses the blood–brain barrier when applied outside the CNS. Several studies in mice (e.g., Vecsernyés etal., 2014; Calias, 2017) or cats (Ward et al., 2010) reported on reduced HPßCD concentrations in the CNS due to its restricted passage through the blood–brain barrier. Effective CNS concentrations can beachieved either by intrathecal administration, or by high intravenously or intraperitoneally applied doses (>4.000 mg/kg), which in turn, can lead to serious side effects such as hearing loss (Davidson etal., 2009; Ward etal., 2010). In addition, a single dose of intraperitoneally administered HPßCD (4.000 mg/kg) has been reported to lead to enhanced development of oligodendrocytes in mice, the loss of which is responsible for demyelination in Npc1 (Kunkel etal., 2023). In humans, a phase 1–2 trial including a very limited number of NPC1 patients with intrathecally administered HPßCD showed a slowed disease progress (Ory etal., 2017). More recently, a phase 1-trial suggests that even intravenously applied HPßCD (1,500 mg/kg or 2,500 mg/kg HPβCD i.v., every 2 weeks) leads to a pharmacological effect in the CNS, even at low concentrations in the cerebrospinal fluid (Hastings et al., 2022). Taking all results together, MIGLU treatment had the smallest negative influence on fresh volume and heights of brain regions and areas of Npc1 +/+ , whereas MIGLU was the therapeutically most beneficial of all tested drugs on fresh volume and heights of brain regions and areas in Npc1 −/− mice. When all structures were examined, the HPßCD treatment had the most negative effect in Npc1 +/+ , whereas it was the therapeutically least beneficial of all tested drugs on fresh volume and heights of brain regions and areas in Npc1−/− mice. The COMBI treatment displayed effects in between those of the MIGLU and HPßCD treatments. Thus, it can bespeculated that the outcome of COMBI treatment is a combination of the more positive effects of MIGLU and the less positive effects of HPßCD treatment. 3.4.4 The drug-induced increase in fresh volume in Npc1−/− mice and functional benefits are partially related A therapeutic approach using the substrate-reduction therapy (MIGLU) and/or the byproduct therapy (HPßCD) has been shown to ameliorate the disease course in Npc1−/− mice (Davidson etal., 2009; Hovakimyan et al., 2013a) and humans (Lachmann et al., 2004; Patterson etal., 2007; Pineda etal., 2009; Brand etal., 2015; SantosLozano etal., 2015), explaining the benefit by the interference of the drugs with the lysosomal cholesterol traffic that is altered by the loss of function of the mutated Npc1 protein (Xie etal., 1999; Zervas etal., 2001; Aqul etal., 2011). However, literature data and own results reveal that the treatment-related benefits on different behaviors vary considerably (Supplementary Table S2). 3.4.5 Drug effects on motor system components in Npc1+/+ and Npc1−/− mice Npc1 +/+ mice: As far as has been studied, no effects of COMBI and MIGLU treatment of Npc1+/+ mice on motor performance can beseen in the accelerod test, walking and swimming speeds in the elevated plus maze test, the open field test and the water maze test (Schlegel etal., 2016). Npc1 −/− mice: As already mentioned by Hung etal. (2016) and Santiago-Mujica etal. (2019), the distance traveled on the rotarod by Npc1−/− mice was significantly less than by Npc1+/+ mice. Concerning drug-induced increase of fresh volume, especially in female Npc1 −/− mice, COMBI, MIGLU, and HPßCD treatment positively influenced spontaneous locomotor abilities and coordination in mutant mice: the total distance and number of visits significantly increased, and accelerod test performance improved with respect to the maximum speed reached (Holzmann et al., 2021). These significant improvements in spontaneous locomotor abilities in COMBI-treated Npc1−/− mice may, at least in part, bethe functional consequence of the drug-induced absolute (Figure 5A) and relative (Figure 9B) increases in white matter volumes, although the respective cortices that form the origin of the callosal fibers did not change their volumes (Figures 3B, 13B). Interestingly, the volume of CPu + GP + ic in MIGLU-treated Npc1 −/− mice significantly exceeded the volume of Sham-treated Npc1 −/− mice (Figure4B). In line with the drug-induced motor improvement was also less cerebellar neurodegeneration after COMBI therapy in Npc1−/− mice (Maass etal., 2015). Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 18 frontiersin.org FIGURE13 (A–D) Relative changes of fresh volume and heights of brain regions and areas of Npc1+/+ and Npc1−/− mice of the COMBI, MIGLU, and HPßCD treatment groups compared with their respective Sham treatment group, calculated as (A) (Npc1+/+ COMBI minus Npc1+/+ Sham)/Npc1+/+ Sham, (B) (Npc1−/− COMBI minus Npc1−/− Sham)/Npc1−/− Sham, (C) (Npc1+/+ MIGLU minus Npc1+/+ Sham)/Npc1+/+ Sham, (D) (Npc1−/− MIGLU minus Npc1−/− Sham)/Npc1−/− Sham, (E) (Npc1+/+ HPßCD minus Npc1+/+ Sham)/Npc1+/+ Sham, (F) (Npc1−/− HPßCD minus Npc1−/− Sham)/Npc1−/− Sham. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 19 frontiersin.org 3.4.6 Drug effects on frontal cortices in Npc1+/+ and Npc1−/− mice Npc1+/+ mice: Total walking distance and relative center distance in the open field test are linked to explorative locomotor activity and anxiety-related behavior. Both COMBIand MIGLU-treated wild type mice showed enhanced anxiety-related behavior in the elevated plus maze compared to Sham-treated wild type mice (Schlegel etal., 2016). The reduced fresh volumes in the frontal cortices of the respective Npc1 +/+ mice could apparently be related to these drug-induced behavioral changes. Npc1 −/− mice: In Npc1 −/− mice, neither treatment resulted in significant changes in anxiety-related behavior, as evidenced by only non-significant differences in relative center times in the open field tests (Holzmann etal., 2021). This non-improvement of the various therapies in Npc1−/− mice are apparently consistent with the unaffected fresh volumes in the hippocampal region (Figure3E) and the frontal cortices (Figures3A, 13B,D,F). 3.4.7 Drug effects on hippocampal areas in Npc1+/+ and Npc1−/− mice Npc1 +/+ mice: The fresh volumes of hippocampal subareas were not changed by either drug treatment (Figures3E, 7A–F). As seen in the water maze test, combination-treated mice performed just as well as Sham-treated wild-types, whereas mice treated with MIGLU displayed impaired spatial learning (Schlegel etal., 2016). Npc1 −/− mice: Compared to the results in the water maze test of Sham-treated Npc1 +/+ mice, the Sham-treated mutants exhibited impairment in remembering the location of the hidden platform. The spatial learning of the mutants, however, did not benefit from drug therapy (Hovakimyan etal., 2013a). Related to this result, the volume of the hippocampal regions was unaltered by COMBI, MIGLU and HPßCD therapy (Figure3E), although some sub-regions showed increased volume in COMBIand/or MIGLU-treated Npc1 −/− mice (Figures7C–E). 3.4.8 Drug effects in olfactory areas and olfaction in Npc1+/+ and Npc1−/− mice Measurements of olfactory-relevant structures in the brain have been carried out, as an impaired sense of smell can bea very early sign of neurodegeneration; this has been demonstrated for Parkinson’s and Alzheimer’s disease (Doty etal., 1987; Hawkes etal., 2009). However, olfactory impairment in NPC1 has not yet been reported in humans. The animal model described in this study shows morphological evidence for dysosmia as seen mainly by a reduced volume of olfactory-related structures in Npc1 −/− mice (see Table 1). In a behavioral buried pellet test, Npc1 mutant mice exposed a significant olfactory deterioration, compared to wild type controls (Meyer etal., 2018). When Npc1 −/− mice were treated with COMBI or HPßCD, we observed an improvement in olfactory performance in the behavioral test and at the same time morphologically a significant reduction in astrogliosis and microgliosis in the olfactory bulb. Also, the BAX/Bcl2 ratio, an indicator of susceptibility to apoptosis in the olfactory bulb, could besignificantly reduced upon treatment, most effectively with HPßCD (Meyer etal., 2018). Depletion of sulfatide lipid [ST (18:1/24:1)] (Figure10), involved in myelin-synthesis, in the internal plexiform layer of the olfactory bulb and the beginning lateral olfactory tract as seen in MSLI may also contribute to Npc1-related olfactory impairment in mice (Meyer etal., 2017). Similar myelination effects of the lateral olfactory tract were reported in Kallmann syndrome in humans (Garcia-Gonzalez etal., 2013). Wewere able to show a significant reduction in the volumes of the olfactory areas in the Npc1 −/− animals compared with the measured values of healthy animals, with particular emphasis on the olfactory bulb and the aforementioned pathways. Generally, dysosmia can also bea result of peripheral events, namely the reduction of olfactory sensory neurons that reach the olfactory bulb only at a severely limited number, as reported in previous reports of our group (Hovakimyan etal., 2013b; Meyer etal., 2018; Witt etal., 2018). However, the number of tyrosine hydroxylaseexpressing interneurons in the olfactory bulb did not change in mutant mice compared to wild type animals (Meyer etal., 2018), and mRNA levels for olfactory marker protein and distinct olfactory receptors remained unchanged speaking mainly for a central reason for olfactory impairment. Therefore, we believe that olfactory dysfunction in Npc1 mice is due to a central transduction problem of sensory inputs that could be represented by the highly reduced myelination levels in tertiary olfactory structures, e.g., the anterior commissure (Table1). Monotherapy with HPßCD had positive effects on body and brain weight as well as on the volume reduction of olfactory areas compared with the Sham therapy. The COMBI therapy was also able to compensate for the loss of body weight and the loss of volume in some olfactory areas. Volume reductions in Sham-treated Npc1 −/− mice were most prominent in structures that relate to high myelinization (anterior commissure, lateral olfactory tract), but were also significant in the piriform cortex and olfactory bulb (Table 1; Figure 11A). However, similar volume changes were also recognizable in the habenula nuclei and the diameters of the commissural pathways anterior commissure and corpus callosum, so that it is not possible to speak of a specifically increased involvement of olfactory structures. In summary, we may conclude that the Npc1 mutations exert a relevant, but not specific, influence on the volumes of olfactory areas. This leads to a reduction in volume, which can bebest ameliorated, but not normalized, by the administration of HPßCD or MIGLU in monotherapy. 4 Conclusion and future research directions The present results show the fresh volumes of brain regions of whole brains and selected areas of Sham-treated Npc1+/+ and Npc1−/− mice as well as data after COMBI, MIGLU and HPßCD treatments of both Npc1 +/+ and Npc1 −/− mice. As there are no causal treatment options, only symptomatic treatments are currently available. Combination therapy with miglustat, HPßCD and allopregnanolone has largely reduced the development of neurological symptoms (Davidson etal., 2009; Meyer etal., 2017, 2018) and also improved visceral symptoms such as hepatoand splenomegaly, but increased both lipolysis and cholesterol transport via abca1 and apoE (Ebner et al., 2018). Side effects of high-dose HPßCD, which lead to degeneration of outer hair cells (Ward etal., 2010; Crumling etal., 2012), must also betaken into account. Since drug-induced changes in the fresh volume of certain brain regions or areas in Npc1 −/− mice could not beclearly linked to behavioral changes, further parameters Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 20 frontiersin.org such as the lipid and protein and receptor composition of the brain need to beinvestigated in the future. In particular, the densities of different lipids are altered differently before and after the non-functional Npc1 protein. In future studies, wewill investigate different parts of the brain of Npc1 +/+ and Npc1 −/− mice by mass spectrometric lipid imaging of representatives of, e.g., STs, gangliosides, glycerolipids, glycerophospholipids, phosphatidic acids and phosphatidylcholines. 5 Materials and methods 5.1 Animals All animal procedures were approved by the local authorities (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei des Landes Mecklenburg-Vorpommern; approval ID: 7221.3–1.1030/12, 14 June 2012). All institutional guidelines for animal welfare and experimental conduct were followed, and all efforts were made to minimize suffering. Heterozygous Npc1+/− mice breeding pairs of Npc1 mice (BALB/ cNctr-Npc1 m1N /−J) were obtained from Jackson Laboratories (Bar Harbor, ME, United States) for generating homozygous Npc1 −/− mutants and Npc1 +/+ control wild type mice. Mice were maintained under standard conditions with free access to food and water with a 12 hday/night cycle, a temperature of 22°C, and a relative humidity of about 60%. Genotypes were determined until postnatal day P7 by PCR analysis of tail DNA (Hovakimyan etal., 2013b; Witt etal., 2018). Npc1 −/− mutants and Npc1 +/+ wild type controls of both sexes were used for different therapeutic treatment schedules. Altogether, 22 wild type and 25 mutant mice of both sexes were involved in this study. The exact numbers of animals investigated in the various groups were: Sham Npc1+/+ 8; COMBI Npc1+/+ 6; MIGLU Npc1 +/+ 1; HPßCD Npc1 +/+ 7; Sham Npc1 −/− 9; COMBI Npc1 −/− 6; MIGLU Npc1 −/− 4; HPßCD Npc1 −/− 6. Brains of both genders were evaluated as there are no significant gender-specific differences in the brain weight (mean ± SD) of sham-treated mice Npc1 +/+ mice (males 0.467 ± 0.015 vs. females 0.463 ± 0.009, p = 0.478) nor in the respective Npc1 −/− mice (males 0.387 ± 0.012 vs. females 0.394 ± 0.016 ±, p = 0.181) (data from Holzmann etal., 2021). 5.2 Treatment We used 4 treatment groups of Npc1 +/+ and NPC1 −/− mice (Figure 14). Combination therapy (COMBI group): Starting at postnatal day 7 (P7) and weekly thereafter, mice were injected with HPßCD/ALLO (25 mg/kg ALLO dissolved in 40% HPßCD, i.p.) (both from Sigma-Aldrich, Munich, Germany). Additionally, from P10 until P23 mice were injected daily with MIGLU (300 mg/kg, i. p.; Zavesca; Actelion Pharmaceuticals, San Francisco, CA), dissolved in saline. Starting at P23 and until termination of the experiment mice were fed standard chow including MIGLU with a daily dose of 1,200 mg/kg. HPßCD monotherapy (HPßCD group): HPßCD was injected starting at postnatal day 7 (P7) and weekly thereafter, in the same amount as included in COMBI (4,000 mg/kg, i. p.; Sigma Aldrich, Munich, Germany). MIGLU monotherapy (MIGLU group): comparable with COMBI, mice were injected daily with MIGLU (300 mg/kg, i. p.) at P10 until P23. From P23 onward, animals were fed standard chow including MIGLU with a daily dose of 1,200 mg/kg. Sham-treated mice (Sham group) were injected following the scheme of the COMBI mice, however, omitting the drugs in the saline. All mice were sacrificed at P65. 5.3 Staining procedures After sacrifice, animals were perfused transcardially via the left ventricle with 15 mL of with 0.9% sodium chloride, followed by 50 mL of Bodian’s fixans, the solution consisting of 900 mL of 80% isopropanol, 50 mL of 37% formaldehyde and 50 mL of glacial acetic acid (Zilles, 1985; Mulisch and Welsch, 2015). Following dissection, brains were weighted and postfixed in the same fixans for 2 days, and subsequently embedded in paraffin wax using standard procedure (Zilles, 1985). Paraffin-embedded specimens, with a section thickness of 20 μm, were used for the Nissl staining (Zilles, 1985). Myelin staining according to Gallyas (1979) was performed on unfixed cryosections, with a section thickness of 20 μm. 5.4 Determination of regional fresh volumes Fresh volumes of specific brain regions were calculated from the volumes determined from paraffin embedded brain slices. Although calculating volumes directly from sections of fixed brain could give reasonable results, wecalculated fresh volumes of brain structures using individual brain-specific shrinkage correction. Shrinkage corrections lead to more accurate measured values. Wefound that working with the uncorrected “raw values” from fixed tissue led to slightly worse p-values in the post-hoc analyses. The delineations of cortical area and brain stem nuclei were based on the criteria given by Wree etal. (1981) and Franklin and Paxinos (2008). The delineations of the 13 brain differentiated regions are illustrated in Supplementary Figures S1–S22. For each structure, the outermost rostral and caudal borders were taken as landmarks. Approximately 10 equidistant sections of the structure of interest located between the landmarks were evaluated (Zilles etal., 1982; Beck etal., 1993). The outlines of the brain areas and layers were traced on paper with the aid of a drawing tube (magnification 15x or 64x), and the area of each structure was determined planimetrically using an image analysis system. For interindividual comparisons it is essential to have a parameter that is independent of the unavoidable shrinkage due to histological procedures. The fresh volume of a brain region was estimated using the Cavalieri method (Gundersen and Jensen, 1987), taking into account the shrinkage factor (for the method, see Wingert, 1969; Kretschmann and Wingert, 1971; Zilles, 1978; Wree etal., 1981). By definition, the shrinkage factor, i.e., the volume of the whole brain after sectioning divided by the fresh volume of the whole brain had to bedetermined for each individual brain separately. Since other investigations (Kretschmann etal., 1975) and our own unpublished results have shown that the weight and the specific weight of fixed brains do not differ from the weight of fresh brains, the fresh volume (FV) of the whole brain can beeasily calculated by weighing the fixed brains and multiplying the brain weight by its specific weight (1.033 g/mL). This value for freshly dissected brains was published by Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 21 frontiersin.org Johansson and Linder (1982) for the rat, by Wingert (1969) for the mouse, and by Zilles (1978) for tupaia, and for fixed human brain by Kretschmann etal. (1982). After sectioning the brain, the total volume of the sectioned brain (SV) has to becalculated from the thickness of the sections (ST), the number of planimetered sections (N), the number of sections between two planimetered sections (n) and the area A of the section iaccording to the following Equation (1): SV nxST xAi i N =+ () = ∑ 1 1 (1) The shrinkage factor (SF) is SV divided by FV (SF = SV/FV). Basically, the determination of the fresh volume of a given brain structure follows the same mathematical procedure as given above. The fresh volume of a structure is Equation (2): F Vn xSTx Ai magnificationxSF i N =+ () = ∑ 1 1 2 (2) 5.5 Data analysis The results are presented as means ± SEM. In all cases, p≤ 0.05 were considered significant. All data were subjected to threeor two-way ANOVA. The Holm-Sidak approach was used for post hoc comparisons. All statistical analyses were done using SigmaPlot 14 Software (Systat Software, Inc., San Jose, CA 95110, UnitedStates). 5.6 Mass spectrometric lipid imaging Mass spectrometric lipid imaging of mouse brain specimens was performed according to Gonzalez de San Roman etal. (2018). Frozen and dried 10 μm sections were coated with matrix (20 mg/mL 2, 5-dihidroxybenzoic acid in 50% ethanol) and analyzed using highmass resolution MALDI-MS in positive and negative ion mode at 100 or 30 μm lateral resolution. According to O’Donnell etal. (2020) wehave referred to all the points such as resolution, ionization mode, matrix and others for mass spectrometric imaging. First, raw data acquired with a MALDI-LTQOrbitrap XL instrument (Thermo) were converted into the public imzML format suitable for SCiLS lab 2018b (Bruker, Bremen, Germany) and normalized using the total ion count (TIC). The regions of interest (ROI) were selected based on the Nissl staining. A receiver-operator characteristic (ROC) analysis was performed for each ROI to determine significantly increased or decreased m/z values in Npc1 −/− animals compared with wild types. The ROC analysis results were then evaluated to find m/z values that were consistently significantly increased or decreased in each ROI across all seven datasets. Distribution maps of these selected m/z values were generated using the SCiLS lab MVS software package with edgepreserving weak image denoising. Distribution fingerprint plots were created with Excel. Lipid species were assigned by a comparison of the measured molecular masses to the Lipid MAPS database, 1 the 1 http://www.lipidmaps.org/ FIGURE14 Timeline of drug administrations for all experimental groups. Mice of the Sham groups were injected with the respective amounts of 0.9% NaCl according to the treatment plan of the combination-treated group. At P7 and thenceforth, NPC1 mice were injected weekly with allopregnanolone (25 mg/kg; Sigma Aldrich, St. Louis, MO) dissolved in HPßCD (4,000 mg/kg, i. p.; Sigma Aldrich). At P10 and until P23, animals were injected daily with miglustat (300 mg/kg, i. p.; Zavesca; Actelion Pharmaceuticals, San Francisco, CA). From P23 onward, animals were fed with miglustat included in standard chow (1,200 mg/kg per day) until termination. Antipova et al. 10.3389/fnana.2024.1430790 Frontiers in Neuroanatomy 22 frontiersin.org Madison Metabolomics database (Metabolomics Society: Databases)2 and previous reports (Berry etal., 2011; Fernandez etal., 2016). For assignment, a maximum of 5 ppm deviation between measured and theoretical mass was selected as the tolerance window. Data availability statement The raw data supporting the conclusions of this article will bemade available by the authors, without undue reservation. Ethics statement The animal study was approved by Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei des Landes MecklenburgVorpommern; approval ID: 7221.3–1.1-030/12, 14 June 2012. The study was conducted in accordance with the local legislation and institutional requirements. Author contributions VA: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing. DH: Investigation, Writing – review & editing. KS: Investigation, Writing – review & editing. JS: Investigation, Writing – review & editing. OS: Formal analysis, Investigation, Methodology, Software, Writing – review & editing. CH: Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. AR: Funding acquisition, Resources, Writing – review & editing. H-JB: Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. EG: Investigation, Methodology, Resources, Software, Writing – review & editing. PH: Investigation, Methodology, Software, Writing – review & editing. KA: Funding acquisition, Investigation, Methodology, Resources, Writing – review & editing. JK: Visualization, Writing – original draft, Writing – review & editing. NH: Resources, Writing – review & editing. MW: Conceptualization, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. AW: Conceptualization, Funding acquisition, Investigation, 2 internationalmetabolomicssociety.org Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing. Funding The author(s) declare financial support was received for the research, authorship, and/or publication of this article This study was supported by the Center of Transdisciplinary Neuroscience Rostock and internal funding as well as by DFG (AM118/7-1). Acknowledgments We would also like to sincerely thank Susann Lehmann, Ulf Haase and Mathias Lietz for their affectionate care of the animals and Frauke Winzer for her excellent histological work (Institute of Anatomy, Rostock). The artwork of Sandra Maria Pietras (Division of Macroscopic and Clinical Anatomy, Medical University of Graz) is gratefully acknowledged. The authors thank Actelion Pharmaceuticals (Allschwil, Switzerland) for the gift of miglustat for the experiments. 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