TDP-43 pathology in the basal forebrain and hypothalamus of patients with amyotrophic lateral sclerosis

Abstract

Introduction: Amyotrophic lateral sclerosis is a neurodegenerative disease characterized clinically by motor symptoms including limb weakness, dysarthria, dysphagia, and respiratory compromise, and pathologically by inclusions of transactive response DNA-binding protein 43 kDa (TDP-43). Patients with amyotrophic lateral sclerosis also may demonstrate non-motor symptoms and signs of autonomic and energy dysfunction as hypermetabolism and weight loss that suggest the possibility of pathology in the forebrain, including hypothalamus. However, this region has received little investigation in amyotrophic lateral sclerosis. In this study, the frequency, topography, and clinical associations of TDP-43 inclusion pathology in the basal forebrain and hypothalamus were examined in 33 patients with amyotrophic lateral sclerosis: 25 men and 8 women; mean age at death of 62.7 years, median disease duration of 3.1 years (range of 1.3 to 9.8 years).

Results: TDP-43 pathology was present in 11 patients (33.3%), including components in both basal forebrain (n=10) and hypothalamus (n=7). This pathology was associated with non-motor system TDP-43 pathology (Χ2=17.5, p=0.00003) and bulbar symptoms at onset (Χ2=4.04, p=0.044), but not age or disease duration. Furthermore, TDP-43 pathology in the lateral hypothalamic area was associated with reduced body mass index (W=11, p=0.023).

Conclusions: This is the first systematic demonstration of pathologic involvement of the basal forebrain and hypothalamus in amyotrophic lateral sclerosis. Furthermore, the findings suggest that involvement of the basal forebrain and hypothalamus has significant phenotypic associations in amyotrophic lateral sclerosis, including site of symptom onset, as well as deficits in energy metabolism with loss of body mass index.

Figure 1

Schematic demonstrating anatomic structures investigated in the basal forebrain (A), mid-hypothalamus (tuberal level) (B), and caudal hypothalamus (mammillary level) (C) (see Materials and methods for detail). To the left of each schematic, grayscale renderings of TDP-43-stained sections of study patients are shown for comparison. In A, the continuity of rostral/chiasmatic hypothalamus and basal forebrain, as discussed in the text, is shown on both the histologic image (which also includes the optic chiasm) and on the schematic (POA). Abbreviations not defined in text: ac – anterior commissure, cc – crus cerebri, fmp – fasciculus mammillaris princeps (mammillary efferents), fx – fornix, GPe/i – external/internal segments of the globus pallidus, FPut – fundus of the putamen, ic – internal capsule, LV – lateral ventricle, mfb - medial forebrain bundle, mmt – mammillothalamic tract, mmtg – mammillotegmental tract, ot – optic tract, 3v – third ventricle.

Figure 2

Basal forebrain pathology in ALS including TDP-43 immunostain examples from six different ALS patients. (A) Representative low-power histologic section (LFB/PAS-hematoxylin) demonstrating magnocellular neurons within nucleus of the diagnonal band (NDB) (pial surface at bottom left of image). (B) Skein-like TDP-43 inclusion within the horizontal limb of the NDB (400 ×, scale bar is 20 microns). (C) Representative lower-power histologic section demonstrating the location of the bed nucleus of the stria terminalis (BNST) and adjacent lateral septal area (LSA) at the level of the internal capsule (ic), anterior commissure (ac), and lateral ventricle (lv) (compare to Figure 1A). (D) Numerous NCIs within the BNST (highlighted by black arrows) (200 ×, scale bar is 50 microns). (E) Low-power LFB/PAS-H histologic section demonstrating ventral striatum (Vs/Is) including clusters or “islands” of small neurons (bottom center of image). (F) Numerous NCIs in small neurons of the VS/Is (400 ×). (G,H) Medium-sized neurons of “substantia innominata” with frequent TDP-43 NCIs and neurities (bottom left of H). These cells could not be attributed to a specific nuclear group or region on H&E stain (see text) (400 ×) (H, inset). A glial cytoplasmic inclusion (at 400 ×) is shown for comparison of cell size with small- and medium-sized neurons with NCIs in panels D, F, G, and H.

Figure 3

Hypothalamic pathology in ALS including TDP-43 immunostain examples from six different ALS patients (five pathologic, one normal). (A) Representative LFB/PAS-hematoxylin-stained section at intermediate magnification demonstrating large neurons and myelinated fiber bundle within the lateral hypothalamic area (LHA). (B) NCIs in the LHA of two different patients (both at 400 ×, scale bar is 20 microns), including a patient with dense, filamentous inclusions (inset). (C) Representative LFB/PAS-hematoxylin-stained section at low power magnification demonstrating the medial mammillary nucleus (MMN) of the mammillary body and the fasciculus mammillaris princeps (fmp), representing the common origin of the mammillotegmental and mammillothalamic tracts. (D) Numerous NCIs (black arrows) and rare GCIs (white arrow) within the MMN (200 ×, scale bar is 50 microns). (E) NCIs within the lateral tuberal nucleus (200 ×) and NCIs, including many granular preinclusions, within ventromedial hypothalamus at the tuberal level (400 ×) (F). (G) Characteristic low power magnification appearance on LFB/PAS-hematoxylin-stained section of large, loosely cohesive neurons in the supraoptic nucleus (SON) overlying the optic tract (opt). (H) SON neurons (seen here at 100 ×, scale bar is 100 microns) did not demonstrate TDP-43 pathology and inclusions were likewise very infrequent in the large, secretory neurons of the paraventricular nucleus (not shown).

Figure 4

Boxplots of BMI relative to involvement of any region in the basal forebrain and/or hypothalamus (left) versus LHA (right) for patients with a BMI measure within ≤ 24 months the time of death (median interval of 3.6 months). Plots represent BMI for basal forebrain/hypothalamus (n = 29; 19 patients without TDP-43 pathology and 10 with inclusion pathology) and for patients with the LHA sampled (n = 25; 21 patients without TDP-43 pathology and 4 with inclusion pathology). There was no significant difference in BMI for patients with and without TDP-43 pathology in any component of forebrain/hypothalamus (left). For the LHA, a significant difference in BMI was identified between patients with and without TDP-43 pathology (p = 0.02). Boxplot prepared in R (R Foundation for Statistical Computing, Vienna, Austria, 2014).

Figure 5

Canonical and non-canonical pathology outside forebrain/hypothalamus in an ALS patient with forebrain/hypothalamic TDP-43 pathology, bulbar symptom onset, and survival of 29.8 months. Pathology in canonical regions, such as lower motor neurons of the hypoglossal nucleus (A), was characterized by neuronal loss, gliosis (white arrows), rarefaction of neuropil, and on TDP-43 immunostain (B), pleomorphic inclusions. Non-canonical TDP-43 pathology was seen in the deep layers of entorhinal cortex (C, D; black arrows indicate neuronal cytoplasmic inclusions in all panels), basolateral amygdala immediately overlying the temporal horn of the lateral ventricle (E, F), and hippocampal sector CA1 (G). This patient also demonstrated glial cytoplasmic inclusions (H, white arrows). All images taken at 200× (scale bar of 50 microns as shown in panel A applies to all panels).