Brain alterations in Lafora disease mouse models

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Fig. 1: Outline of LD. LD, a neurodegenerative epilepsy characterized by the presence of LBs, is an autosomic recessive disease caused by loss-of-function mutations in the genes encoding laforin or malin. Laforin and malin form a complex which modulates intracellular proteolytic systems and regulates, through UPS and GLUTs transport, glycogen synthesis. In addition, laforin is able to dephosphorylate glycogen. LBs are composed of proteins and polyglucosans, which are a low branched, hyperphosphorylated and insoluble form of glycogen. The three proposed pathogenic mechanisms for LD development are the following: 1) the complex dysfunction causes alterations in the intracellular proteolytic systems (a) and 2) deregulation in glycogen synthesis (b, b’), leading to the formation of LBs (a1, b1) and to the clinical traits of LD (d, e). 3) The dysfunction in the glucan phosphatase activity of laforin contributes to the formation of LBs (c).

Fig. 2: Intracellular proteolytic systems (autophagy and UPS) and proposed mechanisms of regulation/modulation by laforin and malin. A) There are three types of autophagy, a lysosome- mediated proteolysis: macroautophagy –a double membrane engulfs cytoplasmic components, including organules, forming the autophagosome which is fused with lysosomes–, microautophagy – lysosomes endocyte cytoplasmic components– and, chaperone-mediated autophagy. –chaperones target substrates which are introduced into the lysososmes through specific channels–. B) Macroautophagy detail of square from A, showing that proteins to be degraded are targeted with poly- ubiquitin chains, which are recognized by p62, leading to macroautophagy degradation. LC3-II marks autophagosome membranes. C) UPS: Proteins are targeted with poly-ubiquitin chains to be degraded by the proteasome. Ubiquitin is linked at protein by an E3 ubiquitin-ligase. E1 and E2 are the responsible for attach the ubiquitin to the E3. Ubiquitins are reused. Images from Muzushima N and Komatsu M 2011 (autophagy) and from Boston Biochem (UPS). D) Proposed mechanisms of laforin and malin in the regulation/modulation of the intracellular proteolytic systems. Laforin and malin form a complex, which interaction is regulated by AMPK, that ubiquitinates proteins to be degraded by proteasome or autophagy. In addition, laforin (by mTOR pathway) and the laforin-malin complex modulate macroautophagy.

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Fig. 3: Epileptogenesis is the process which leads to epilepsy (and chronic epilepsy), a disorder characterized by an enduring predisposition to generate epileptic seizures. Epilepsy could develop as a consequence of different triggering events. There are functional and structural alterations during epileptogenesis involving abnormal synchronization of the circuits and/or excitation/inhibition unbalance. Other alterations not related to neuronal function could also accompany. Colored squared represent the progression of epileptogenesis: yellow is the onset of the epileptogenic process in which a predisposition to generate seizures increases; orange represents the first spontaneous seizure, which together with a high recurrent risk (increased predisposition) marks the onset of clinical epilepsy;

brown represent the progression towards chronic epilepsy, in which predisposition is the highest and alterations make seizure recurrent, severe and often pharmaco-resistant.

Fig. 4: Time dependent accumulation of LBs in the brain of MKO mice. Sagittal sections of different brain regions (as indicated in the panels) from 3 and 5 months-old MKO mice, and LKO and WT mice at 5 months of age. Sections were stained with PAS and counterstained with hematoxylin. Note that MKO sections present a time dependent accumulation of LBs as observed in LKO at 5 months. LBs are absent in WT mice, in which PAS-staining is only occasionally observed around blood vessels (arrow). CA1, Cornus Ammonis 1; ml, molecular layer; Pcl, Purkinje cell layer; gl, granule cell layer. Scale bar, 30μm.

Fig. 5: Differential accumulation of LBs between the pontine nuclei and cortex of MKO and LKO mice at early adult stages. Sagittal sections of different brain regions (as indicated in panels) from 2-3 months- old MKO and LKO mice stained with PAS and counterstained with hematoxylin. Note that MKO had more LBs in pontine nuclei (A) and fewer in the cortex (C) than LKO mice (B, D). Scale bar, 30 μm.

Fig. 6: Schematic spatio-temporal representation of LBs accumulation in MKO and LKO brains. Drawings represent sagittal sections of the brain (modified from Allen Database Atlas). Red dots represent LBs. As indicated in the panels, there is a time dependent differential accumulation between the two KO mice before 5 months of age. Afterwards, the entire brain is covered with LBs. OB, Olfactory Bulb; OBN, Olfactory Bulb Nucleus; Cx, cortex; Hp, hippocampus; Coll, inferior and superior colliculi; Cb, cerebellum;

PN, Pontine Nuclei; PrON, preoptic nucleus; Thal, thalamus.

Fig. 7: Laforin levels increase in MKO P16 brain, and translocate from the soluble to the insoluble fraction with age. A) Western-blot analysis of laforin in total extracts of brainstem (Bs), cortex (Cs) and hippocampus (Hp) of P16 WT, LKO and MKO mice. Note the accumulation of laforin in MKO mice. Laforin (B) and MGS (C) levels from P16 and 3 months-old MKO mice. Total (T), soluble (S) and insoluble (P) fractions. Note that both laforin and MGS are found predominantly in the soluble fraction at P16 but colocalized to the insoluble fraction at 3 months of age. The levels of -tubulin were used as a loading control.

Fig. 8: Immunolocalization of laforin uncovers LBs heterogeneity. Thin consecutive sections (10μm) from 3.5 months-old hippocampus (A, B) and heart (D, E) MKO mice were stained with PAS and hematoxylin (A, D). Laforin immunostaining (B, E) shows a similar pattern and highlights the presence of two types of structures. Arrowheads mark large round aggregates and thin arrows mark small irregular ones.

Double staining with laforin immunohistochemistry and PAS staining in the hippocampus (C) and heart (F) from 3.5m MKO mice show colocalization of laforin in LBs. Scale bar 30 μm (A-E) and 50 μm (F).

Fig. 9: Laforin accumulates in LBs. Electron microscopic panoramic view from 5 months-old MKO cerebellum (A) and a high magnification view of the same region (B). C) Laforin immunogold staining (dark dots) in a different section. Yellow asterisks mark LBs. Note the accumulation of gold particles in LB. Scale bar, 4 μm (A), 500nm (B), 250nm (C). Purkinje cell (Pc), granullar cell layer (gcl).

Fig. 10: Molecular heterogeneity of LBs in MKO mice. Confocal z- stack projections (A-F), z-stack slices (G-I) and mid-views (J) of 5 months-old MKO brains co- immunostained with anti- laforin (green) and anti- ubiquitin (Ubq) or anti-glycogen synthase (MGS) , Grp78, p62 (all in red). Note that the small irregular LBs are marked with ubiquitin and Grp78, the large ones with MGS. p62 shows a patchy distribution sometimes overlapping with LBs. Scale bar, 25um (A-F), 10um (G-J).

Fig. 11: Treatment with Rapamycin increases PAS-positive granules in the hippocampus. PAS and hematoxylin stained sagittal sections of the hippocampal region from WT, LKO and MKO mice at 2 months of age, after (6 weeks) treatment with or without Rapamycin (D-I). Note the abundant accumulation of LBs with Rapamycin treatment. CA1, Cornus Ammonis 1. Scale bar, 100 μm.

Fig. 12: Treatment with Rapamycin increases PAS-positive aggregates in the cerebellum. PAS and hematoxylin stained sagittal sections of the cerebellum from WT, LKO and MKO mice at 2 months of age, with or without (6 weeks) Rapamycin treatment (D-I). Note the abundant accumulation of LBs with Rapamycin treatment in mutant mice. ml, molecular layer; Pcl, Purkinje cell layer; gl, granular layer. Scale bar, 100 μm.

Fig. 13: Rapamycin does has little impact in memory performance but differentially affects body weight. A) Discrimination index in the Object Recognition Test of WT, MKO and LKO mice. B) Number of fecal pellets of WT, MKO and LKO.

ANOVA One-way and post hoc Turkey test show statistical differences: p<0.05. The number of tested animals are as follows: WT veh (10), WT rapa (10), MKO veh (5), MKO rapa (5), LKO veh (5), LKO rapa (5). Veh, vehicle treatment;

rapa, rapamycin treatment.

Fig. 14: Glucan phosphatase activity of laforin is dispensable to prevent LB formation. PAS-staining of hippocampal sections from WT, LKO, LKO;Laf^WT and LKO;Laf^C265S at different ages (as show in the panels). Arrowheads mark LBs. CA1, Cornus Ammonis 1. Scale bar, 100 μm.

Table 1: Presence of LBs in the hippocampus. The number of analyzed animals that present no (white), sparse (light gray), intermediate (dark gray) or dense (black) accumulation of LBs.

Fig. 15: Glucan phosphatase activity of laforin is dispensable to prevent LBs formation. PAS-staining of cerebellar sections from WT, LKO, LKO;Laf^WT and LKO;Laf^C265S at different ages (as show in the panels).

Molecular layer (ml), Purkinje cell layer (Pcl) and granular layer (gl). Scale bar, 100 μm.

Fig. 16: Glucan phosphatase activity of laforin is dispensable to prevent LBs formation. PAS-staining of heart sections from WT, LKO, LKO;Laf^WT and LKO;Laf^C265S at different ages (as show in the panels). Scale bar, 100 μm.

Fig. 17: Transgenic expression of laforin with or without glucan phosphatase activity partial rescues the memory performance of LKO mice. Object Recognition Test performed in 7 months-old WT, LKO, LKO;Laf^WT and LKO;Laf^C265S mice. Data are represented using box-plot that shows minimal and maximal values as well as lower quartile, median, upper quartile, and outliers. *, Significant difference with p<0.05.

Each group of analyzed mice was composed of 12-18 animals.

Fig. 18: LBs do not preferentially accumulate in interneurons. Brain sections from 3.5 months-old MKO mice stained with PAS and antibodies against calbindin (A-C). PAS inclusions and calbindin showed partial colocalization in the cortex (A), pontine nuclei (B) and cerebellum (C) whereas PAS inclusions did not colocalize with calretinin (D, E) in the cortex (D) and cerebellum (E). Molecular layer (ml), Purkinje cell layer (Pcl) and granular layer (gl). Scale bar, 10 μm.

Fig. 19: LB-free Purkinje cell degenerate in LKO mice. Electron microscopic images from 5 months-old MKO cerebellum. A) Panoramic view shows two Purkinje cells, one in the process of degeneration (dark one). B) High magnification of the area squares in A shows a LB (yellow asterisk) in a process of a cell close to the degenerating Purkinje cell. Scale bar, 6 μm (A) and 2 μm (B). Purkinje cell layer (Pcl), granular cell layer (gcl).

Fig. 20: Adult LKO and MKO mice present normal number of interneurons. A) Representative images of calbindin (Cb), calretinin (Cr) and PV immunostaining, or SST and VIP hybridization (as show in the panels) in sagittal sections (10 μm) of the motor cortex 1 (M1) form 5 months-old WT, LKO and MKO mice. B) The graph illustrates the quantification of the number of neurons in relative units. Error bars represent the standard deviation. C) The table shows the number of animals analyzed per genotype and interneuron type. ANOVA one-way and post hoc Turkey test do not show significant differences.

Scale bar, 50 μm.

Fig. 21: Dendritic branching of MKO and LKO pyramidal CA1 P16 neurons is increased. The graphs represent Sholl analysis of pyramidal CA1 hippocampal neurons from P16 WT, LKO and MKO mice visualized by DiOlistic labeling and confocal analysis. Apical (A, B) or basal (C, D) dendritic length (A, C) and nº of nodes (B, D) as a function of the distance from the soma center. The number of animals and neurons analyzed per genotype for apical and basal dendritic tree are the following: WT (4, 8 apical; 5, 16 basal), MKO (4, 15; 4, 9), LKO (4, 11; 4, 9). ANOVA one-way and Turkey post hoc show statistical differences: WT vs MKO (*), WT vs LKO (#), MKO vs LKO (+); with p<0.05 (*), p<0.01 (**), p<0.001 (***).

Fig. 22: There is no significant difference in the number of dendrites of the same branching order in LKO and MKO neurons. A) Number of secondary, tertiary, quaternary or higher order apical dendrites. B) Number of primary, secondary, tertiary, quaternary or higher order basal dendrites.

Fig. 23: Spines of LKO and MKO neurons differ from those of WT in opposite directions. A) Representative images of dendritic spines of pyramidal CA1 hippocampal neurons from P16 WT, MKO, LKO, LKO;Laf^C265S and LKO;Laf^WT animals visualized by DiOlistic labeling and confocal studies. B) Representation of the caricatured average spine of each genotype. Quantifications of neck length (C), head diameter (D) and spine density (E). Note that LKO and MKO spines have opposite morphology and that of transgenic mice is closer to that of MKO. Error bars represent standard deviation. Note the higher variability (dynamic morphology) in neck length of LKO and the low variability of MKO and transgenic spines compared to WT ones. At least 4 animals per genotype and ≥3 neurons per animal, for a total of 2100-4800 spines per genotype were analyzed. ANOVA One-way and Turkey post hoc test shows statistical differences among WT (*), MKO (+), LKO (#) and LKO;Laf^C265S (@): p<0.05 (*), p<0.01 (**) and p<0.001 (***). Scale bar, 10 μm.

Fig. 24: Laforin levels are increased in MKO, LKO;Laf^WT and LKO;Laf^C265S in P16 hippocampal synaptosomes. A) Western-blots from P16 hippocampal total lysates and synaptosomal preparations (2 animals per genotype). B) Electron microscopic picture with an example of synaptosome from P16 WT, scale bar 500nm. Laforin quantifications normalized to tubulin and WT levels, in total lysates (C) and synaptosomal preparation (D).

Fig. 25: Laforin localizes to the synapses. Electron microscopic images of synaptosomes from P16 MKO hippocampus or from 5 months-old MKO hippocampus stained with antibodies against laforin followed by immunogold. Note that laforin (black dots) localizes in the pre- and the postsynaptic compartments. Scale bar, 200nm.

Fig. 26: LKO and MKO P16 hippocampal slices have opposite synaptic responses. Electrophysiological field recordings at CA1 in acute brain slices from 16 days-old WT, LKO and MKO mice with and without picrotoxin (Pic). A) Representative traces at 50 μA stimulation with (red line) or without picrotoxin (black line). B) Input-output curve without drug (circles) was higher in LKO slices (p<0.05 from 10-200 μA) and similar in MKO slices, as compared with WT. Picrotoxin (triangles) increased the response in all genotypes: WT and LKO (both, p<0.05 from 10-200 μA), whereas in MKO slices changes were not significant. Excitatory response (curves with picrotoxin) was slightly higher, although no significant, in LKO, and significantly lower in MKO (p<0.05 from 10-300 μA) as compared to WT slices. C) Percentage of slices with epileptic activity observed only in the presence of picrotoxin at different intensities of stimulation. LKO slices were more prone to epileptic activity than WTs whereas MKO present a decreased frequency (p<0.05 from 40-60 μA) as compared to WT. Error bars represent standard error.

ANOVA One-way and Turkey post hoc. The number of animals and total slices used, per each genotype and condition are as following: WT (8, 25), WT+Pic (2, 22), LKO (10, 38), LKO+Pic (5, 21), MKO (7, 26) and MKO+Pic (5, 23).

Fig. 27: Current proposed outline of LD. LD, a neurodegenerative epilepsy characterized by the presence of LBs, is an autosomic recessive disease caused by loss-of-function mutations in the genes encoding laforin or malin. Laforin and malin form a complex which modulates intracellular proteolytic systems – macroautophagy and UPS— and regulates, through UPS and GLUTs transport, glycogen synthesis. In addition, laforin is able to dephosphorylate glycogen. LBs are composed of proteins and polyglucosans, which are a low branched, hyperphosphorylated and insoluble form of glycogen. The current proposed pathogenic mechanisms for LD are the following: The complex dysfunction causes macroautophagy impairment (a), UPS defects (b) and glycogen accumulation due to a deregulation in glycogen synthesis. This upregulation is caused by UPS defects –leading to an accumulation of PTG— (b1) and, by a GS activation through the increased levels of G6P (c1), caused by an increased levels of GLUTs at the plasma membrane because the laforin-malin complex dysfunction (c). The three altered mechanisms –macroautophagy impairment, UPS defects and glycogen synthesis— contribute to the formation of LBs (d1-d3). The deficiency in the glucan phosphatase activity of laforin does not cause LBs formation but could potentiate the LBs maturation (d4). The laforin-malin complex could detect and avoid the development of the structures which become into LBs, thus, the complex dysfunction could potentiate the formation of LBs (d’1, d’2, d’3). Laforin is sequestered in LBs (f). Laforin deficiency, caused by loss-of-function mutations (g1) or by the presence of LBs (f, g2), promotes a deregulation in spine morphology, which could affect to the pathology (e5), and also promotes an increased synaptic transmission, which could lead to epilepsy (e6). The increased neuronal branching due to a dysfunction of the laforin-malin complex (h) could have an impact on LD development (e7).

Macroautophagy impairment, UPS defects and LBs could lead to the pathology (e1, e2, e4, respectively), while glycogen accumulation contributes to neurodegeneration (e3). Neurodegeneration leads to neuronal death, which could promote epilepsy indirectly (i1), whereas epilepsy could cause neuronal death by excitotoxicity (i2).