Loss of Myelin-Associated Glycoprotein in Kearns-Sayre Syndrome

Figure 1. Pattern of myelin loss within the dentate nucleus in Kearns-Sayre syndrome (KSS). There was an apparent loss of myelin-associated glycoprotein (MAG) (A-C) and 2′,3′-cyclic nucleotide phosphodiesterase (CNPase) (D-F) compared with myelin oligodendrocyte glycoprotein (MOG) (G-I) and myelin basic protein (MBP) (J-L) immunoreactivity within the dentate nucleus white matter (KSS patient 1). G-I, The loss of MOG was much less striking than MAG loss in serial sections. At high magnification (original magnification ×63), the loss of MAG (A-C) and CNPase (D-F) in KSS was notable compared with corresponding control tissue. In contrast, the difference in MOG (G-I) and MBP (J-L) immunoreactivity between KSS and control tissue was subtle, if at all. Scale bar represents 12 μm.

Figure 2. Injury to oligodendrocytes, relative sparing of axons, and lack of inflammation within dentate in Kearns-Sayre syndrome (KSS). A-D, Loss of Olig2-positive nuclei showed a notable decrease in oligodendrocyte lineage cells within the white matter of the dentate nucleus in KSS patients (B) compared with controls (A and D). Quantitation of Olig2-positive cells in KSS dentate nucleus white matter demonstrated the loss of oligodendrocytes to be significant (P = .008, analysis of variance) compared with controls and patients with mitochondrial encephalopathy lactic acidosis and strokelike episodes (MELAS) in which myelin-associated glycoprotein (MAG) loss was not detected in the dentate (D). In nondemyelinated regions in KSS cerebrum, we did not detect a significant loss of Olig2-positive cells compared with corresponding white matter. Apoptosis-inducing factor (AIF) was located in the cytoplasm of neurons (C, arrowheads), whereas neuronal nuclei did not show AIF immunoreactivity. In contrast, numerous nuclei with intense AIF staining were detected in the KSS dentate white matter where preferential MAG loss was evident (C, arrows). E-G, Axons in the dentate nucleus white matter, where MAG loss was apparent, and control tissue were morphologically intact as judged by Bielschowsky silver stain. Axons in the MAG loss region rarely showed accumulation of synaptophysin, a marker of fast axonal transport block (G). In contrast, synaptophysin-positive axons were detected within affected regions with equal loss of myelin components in primary mitochondrial disorders, reflecting ongoing axonal degeneration (F, arrowheads). H-J, CD68 immunoreactivity was present at levels comparable to controls (I) in KSS dentate white matter (H), where preferential loss of MAG was apparent. In contrast, CD68-positive cells were abundant in pattern III multiple sclerosis (MS) (n = 5), white matter stroke (WMS) (n = 4), and progressive multifocal leukoencephalopathy (PML) (n = 3) lesions, reported disorders with distal “dying-back” oligodendrogliopathy (J). Error bars indicate SEM.

Figure 3. The single deletion of mitochondrial DNA (mtDNA) within the white matter of the dentate nucleus in Kearns-Sayre syndrome (KSS) is pathogenic. A, A single deletion of mtDNA was detected in laser microdissected regions from KSS dentate nucleus white (lane 1) and gray (lane 2) matter using long-range polymerase chain reaction (PCR); lane 3 shows full-length amplified product from control DNA sample (meninges). B, Real-time PCR revealed significantly greater heteroplasmy levels in dentate white and gray matter (P < .001) than the corresponding regions in control tissue in 3 separate experiments. However, heteroplasmy levels in KSS dentate white matter (WM) were higher than the 60% threshold and significantly greater than in gray matter (GM). In primary mtDNA disorders without myelin-associated glycoprotein loss and due to POLG mutations (3 patients), the heteroplasmy level in dentate white matter was less than 60% and significantly less than in KSS. C, Sequencing of the single, large-scale mtDNA deletion extracted from the long-range PCR gel confirms the 3978–base pair (bp) deletion. The mtDNA deletion removed several key genes and occurs at the site of an imperfect 12-bp repeat (highlighted).

Figure 4. Mitochondrial respiratory chain complex subunit expression and complex IV (COX) activity in Kearns-Sayre syndrome (KSS) dentate white (WM) and gray (GM) matter. A-D, Immunohistochemical detection of mitochondrial proteins within dentate nucleus WM showed comparable density of porin (A) and complex II (SDH) 70-kDa (B) in KSS (Ai and Bi) and controls (Aii and Bii). There was a notable reduction in complex I 30-kDa (C) and complex IV subunit-I or COX-I (D) in KSS (Ci and Di) compared with controls (Cii and Dii). Densitometric analysis showed a significant (P <.001) decrease in complex I 30-kDa (by 30.7%) and COX-I (by 31.4%) immunoreactivity in KSS (mean [SD], 100.3 [4.4] and 79.9 [5.0] for complex I 30-kDa and COX-I, respectively) compared with controls (144.7 [6.8] and 116.4 [8.0] for complex I 30-kDa and COX-I, respectively). E, Immunofluorescent labeling of phosphorylated neurofilament (axons, red), mitochondria (porin, green) and Cox-I (blue) identified a striking loss of COX-I in the nonaxonal population of mitochondria. In contrast, COX-I is relatively spared in axonal mitochondria. F and G, Immunofluorescent labeling of CNPase (oligodendrocytes, red) or glial fibrillary acidic protein (astrocytes, red), mitochondria (porin, green), and COX-I (blue) confirmed the lack of COX-I–positive elements within oligodendrocytes (F) and astrocytes (G). Supplementary eFigure 3 shows COX-I and porin labeling within astrocytes and oligodendrocytes in control tissue. H, COX and succinate dehydrogenase immunohistochemical analysis identified numerous respiratory-deficient cells in the KSS dentate nucleus WM (H), however, such cells were absent in KSS GM and WM from control cases (Supplementary eFigure 3, C-D). I-L, Porin (I), SDH 70-kDa (J), and COX-I (L) immunoreactivity were not diminished within KSS dentate nucleus neurons (Ii, Ji, and Li) compared with controls (Iii, Jii, and Lii). However, complex I 30-kDa was apparently decreased within KSS patient 1 dentate nucleus neurons (Ki) compared with controls (Kii). Scale bar represents 100 μm.