Huntington's disease (HD): degeneration of select nuclei, widespread occurrence of neuronal nuclear and axonal inclusions in the brainstem

Abstract

Huntington's disease (HD) is a progressive polyglutamine disease that leads to a severe striatal and layer-specific neuronal loss in the cerebral neo-and allocortex. As some of the clinical symptoms (eg, oculomotor dysfunctions) suggested a degeneration of select brainstem nuclei, we performed a systematic investigation of the brainstem of eight clinically diagnosed and genetically confirmed HD patients. This post-mortem investigation revealed a consistent neuronal loss in the substantia nigra, pontine nuclei, reticulotegmental nucleus of the pons, superior and inferior olives, in the area of the excitatory burst neurons for horizontal saccades, raphe interpositus nucleus and vestibular nuclei. Immunoreactive intranuclear neuronal inclusions were present in all degenerated and apparently spared brainstem nuclei and immunoreactive axonal inclusions were observed in all brainstem fiber tracts of the HD patients. Degeneration of brainstem nuclei can account for a number of less well-understood clinical HD symptoms (ie, cerebellar, oculomotor and vestibular symptoms), while the formation of axonal aggregates may represent a crucial event in the cascades of pathological events leading to neurodegeneration in HD.

Keywords: Huntington's disease; axonal inclusions; brainstem; neurodegeneration; pathoanatomy; polyglutamine diseases.

Figure 1

The lower brainstem in  H untington's disease ( HD). A. Ventral aspect of the lower brainstem of a typical 76‐year‐old male control individual (control individual 11; Table 3). B. The lower brainstem with a flattened pons (asterisk) of a clinically diagnosed and genetically confirmed female HD patient (onset of HD symptoms: 40 years; age at death: 68 years; CAG‐repeats in the mutated HD allele: 46; Vonsattel grade of striatal atrophy: 3) (HD patient 5; Tables 1 and 2) 81, 82, 83. Abbreviations: CP = cerebral peduncle; IO = inferior olive; MCP = medial cerebellar peduncle; PYR = pyramidal tract; V = trigeminal nerve; VI = abducens nerve; VII = facial nerve; VIII = Vestibulocochlear nerve.

Figure 2

Neuronal loss in mid‐pontine and pontomedullary nuclei in  H untington's disease ( HD ). A. Horizontal section through the mid‐pons of a representative 66‐year‐old male control individual depicting the oculomotor reticulotegmental nucleus of the pons (RTTG) (control individual 10; Table 3). B. Considerable neuronal loss in the RTTG of a 64‐year‐old male HD patient (CAG‐repeats in the mutated HD allele: 41; age at HD onset: 55 years; duration of HD: 9 years; Vonsattel grade of striatal atrophy: 2; patient 3; Tables 1, 2 and 4). Inset shows remaining RTTG nerve cells and lipofuscin deposits in the neuropil (arrow). C. Horizontal section through the caudal pons of a typical 54‐year‐old male control individual depicting the area of the excitatory burst neurons (arrowheads) for horizontal saccades (EBR) with its typical horizontally oriented nerve cells (control individual 5; Table 3). D. Complete neuronal loss in the EBR of a 51‐year‐old male HD patient (CAG‐repeats in the mutated HD allele: 49; age at HD onset: 33 years; duration of HD: 18 years; Vonsattel grade of striatal atrophy: 4; patient 1; Tables 1, 2 and 4). E. Horizontal section through the pontomedullary junction of a 39‐year‐old woman without any medical histories of neuropsychiatric diseases showing the RIP with its typical omnipause neurons for saccades in all directions (arrowheads) (control individual 3; Table 3). F. Severe loss of RIP omnipause neurons of a 70‐year‐old female HD patient. Arrow points to a surviving RIP nerve cell (CAG‐repeats in the mutated HD allele: 44; age at HD onset: 50 years; duration of HD: 20 years; Vonsattel grade of striatal atrophy: 2; patient 7; Tables 1, 2 and 4) (A–F: aldehyde‐fuchsin Darrow red staining; 100 μm PEG sections). Abbreviations: MLF = medial longitudinal fascicle; PNO = pontine reticular nucleus, oral subnucleus; RIP = raphe interpositus nucleus; VI = abducens nerve; VII = facial nerve.

Figure 3

Neuronal loss in the pontomedullary junction and medulla oblongata in  H untington's disease ( HD ). A. Horizontal section through the pontomedullary junction of a typical 54‐year‐old male control individual depicting the lateral (LV) and magnocellular portion of the medial (MV) vestibular nuclei (control individual 5; Table 3). B. Severe loss of LV and MV nerve cells of a 61‐year‐old female HD patient (CAG‐repeats in the mutated HD allele: 45; age at HD onset: 35 years; duration of HD: 26 years; Vonsattel grade of striatal atrophy: 3; patient 2; Tables 1, 2 and 4). Arrow points to a remaining giant LV nerve cell. C. Horizontal section through the mid‐pons of a representative 54‐year‐old male control individual depicting the auditory superior olive (SO) with its medial (MSO) and lateral subnuclei (LSO) as well as its periolivary group (SOP) (control individual 5; Table 3). D. SO neuronal loss of a 61‐year‐old female HD patient (CAG‐repeats in the mutated HD allele: 45; age at HD onset: 35 years; duration of HD: 26 years; Vonsattel grade of striatal atrophy: 3; patient 2; Tables 1, 2 and 4) predominantly affecting the LSO and SOP. E. Horizontal section through the medulla oblongata a 84‐year‐old male control individual without any medical histories of neuropsychiatric diseases showing the inferior olive (IO) with its principal (IOP) and medial accessory subnuclei (IOM) (control individual 13; Table 3). F. Circumscribed severe neuronal loss in the IOP (asterisk) of an 91‐year‐old male HD patient (CAG‐repeats in the mutated HD allele: 42; age at HD onset: 55 years; duration of HD: 36 years; Vonsattel grade of striatal atrophy: 2; patient 8; Tables 1, 2 and 4). Inset shows devastated area of the IOP (marked in F by an asterisk) with severe neuronal loss and the presence of extraneuronal lipofuscin deposits. Arrowheads point to remaining IOP nerve cells and arrows to extraneuronal lipofuscin deposits which can serve as reliable marker for neurodegeneration (A–F: aldehyde‐fuchsin Darrow red staining; 100 μm PEG sections). Abbreviations: IO = inferior olive; IOM = inferior olive, medial accessory subnucleus; IOP = inferior olive, principal subnucleus; LSO = lateral superior olive; LV = lateral vestibular nucleus; MSO = medial superior olive; MV = medial vestibular nucleus, magnocellular portion; SO = superior olive; SOP = superior olive, periolivary group; TZ = trapezoid body; VII = facial nerve.

Figure 4

p62 immunoreactive neuronal intranuclear inclusions in spared and degenerated brainstem nuclei in Huntington's disease (HD). P62 immumopositive neuronal intranuclear inclusions (NI) in well‐preserved brainstem nuclei in HD: A. pedunculopontine nucleus (PPT), B. dorsal raphe nucleus (DR), C. locus coeruleus (LC), D. motor trigeminal nucleus (MOV), E. gigantocellular reticular nucleus (GI), F. dorsal motor vagal nucleus (DMV), G. hypoglossal nucleus (XII), H. external cuneate nucleus (ECU). NI in degenerated brainstem nuclei in HD: I. substantia nigra (SN), J. pontine nuclei (PN), K. reticulotegmental nucleus of the pons (RTTG), L. facial nucleus (VII), M. superior olive (SO), N . medial vestibular nucleus (MV), O. lateral vestibular nucleus (LV), P. inferior olive (IO) (A, B, F, L, N, O: HD patient 1—Table 1, CAG‐repeats in the mutated HD allele: 49; age at HD onset: 33 years; duration of HD: 18 years; Vonsattel grade of striatal atrophy: 4; C, G, H, M: HD patient 7—Table 1, CAG‐repeats in the mutated HD allele: 44; age at HD onset: 50 years; duration of HD: 20 years; Vonsattel grade of striatal atrophy 2: D, I: HD patient 4—Table 1, CAG‐repeats in the mutated HD allele: 45; age at HD onset: 53 years; duration of HD: 12 years; Vonsattel grade of striatal atrophy: 4; E, J, K, P: HD patient 6—Table 1, CAG‐repeats in the mutated HD allele: 40; age at HD onset: 60 years; duration of HD: 8 years; Vonsattel grade of striatal atrophy: 3) (A–P: p62 immunohistochemistry, counterstaining with aldehyde‐fuchsin Darrow red; 100 μm PEG sections).

Figure 5

p62 immunoreactive axonal neuronal inclusions in brainstem fiber tracts in Huntington's disease (HD). P62 immumopositive axonal neuronal inclusions in HD: (A) oculomotor nerve (III), (B) trochlear nerve (IV), (C) pontocerebellar fibers (PCF), (D) PCF), (E) trigeminal nerve (V), (F) facial nerve (VII), (G) trapezoid body (TZ), (H ) vestibulocochlear nerve (VIII), (I) inferior cerebellar peduncle (ICP), (J) intermediate reticular zone (IRZ), (K) olivocerebellar fibers (OCF), (L) hypoglossal nerve (XII), (M) hypoglossal nerve (XII), (N) cuneate fascicle (ECU), (O) solitary tract (SOL), (P) dorsal spinocerebellar tract (DST). (A–P: p62/AT270 double immunohistochemistry; 100 μm PEG sections) (A, E, K, N: HD patient 4—Table 1, CAG‐repeats in the mutated HD allele: 45; age at HD onset: 53 years; duration of HD: 12 years; Vonsattel grade of striatal atrophy: 4; B, L, P: HD patient 8—Table 1, CAG‐repeats in the mutated HD allele: 42; age at HD onset: 55 years; duration of HD: 36 years; Vonsattel grade of striatal atrophy: 2; C, D, F, G, H: HD patient 2—Table 1, CAG‐repeats in the mutated HD allele: 45; age at HD onset: 35 years; duration of HD: 26 years; Vonsattel grade of striatal atrophy 3; I: HD patient 5—Table 1, CAG‐repeats in the mutated HD allele: 46; age at HD onset: 40 years; duration of HD: 28 years; Vonsattel grade of striatal atrophy 3; J, O: HD patient 6—Table 1, CAG‐repeats in the mutated HD allele: 40; age at HD onset: 60 years; duration of HD: 8 years; Vonsattel grade of striatal atrophy: 3; M: HD patient 1—Table 1, CAG‐repeats in the mutated HD allele: 49; age at HD onset: 33 years; duration of HD: 18 years; Vonsattel grade of striatal atrophy: 4).

Figure 6

p62 and 1C2 double immunofluorescence of axonal inclusions in brainstem fiber tracts in Huntington's disease (HD). Double immunofluorescence of intra‐axonal neuronal inclusions in the nigrostriatal pathway (A–C) and (D–F) pontocerebellar fibers of a representative 51‐year‐old male HD patient (CAG‐repeats in the mutated HD allele: 49; age at HD onset: 33 years; duration of HD: 18 years; Vonsattel grade of striatal atrophy: 4; patient 1; Tables 1, 2 and 5). Immunolabeling with (A,D) an antibody against the proteasomal shuttle protein p62 (red) and (B,E) the anti‐polyglutamine marker 1C2 (green). The positive immunoreactions for the anti‐polyglutamine marker 1C2 point to the recruitment of the pathologically altered form of the disease protein huntingtin with an elongated polyglutamine stretch into the p62 immunoreactive axonal inclusions (A, D: p62 immunostaining—Cy3; B, E: anti‐polyglutamine 1C2 immunostaining—Alexa 488; 100 μm PEG sections).

Figure 7

p62 and anti‐ubiquitin double immunofluorescence of axonal inclusions in brainstem fiber tracts in  H untington's disease ( HD ). (A–C) Double immunofluorescence of intra‐axonal neuronal inclusions in the nigrostriatal pathway of a representative 64‐year‐old male HD patient (CAG‐repeats in the mutated HD allele: 41; age at HD onset: 55 years; duration of HD: 9 years; Vonsattel grade of striatal atrophy: 2; patient 3; Tables 1, 2 and 5) immunolabeled with (A) an antibody against the ubiquitin protein (green) and (B) the proteasomale shuttle protein p62 (red). The presence of ubiquitin and p62 in intra‐axonal aggregates (i) suggests an involvement of the ubiquitin‐proteasome pathway in the HD pathogenesis and; (ii) points to defense mechanisms of affected nerve cells that attempt to reduce levels of mutant huntingtin; but (iii) may also reflect a decreased or compromised activity of the ubiquitin‐proteasome pathway or its overload in HD (A: p62 immunostaining—Alexa 488; B: anti‐ubiquitin immunostaining—Cy3; 100 μm PEG sections).