Clinical features
Brief summaries of the demographic and clinical information are provided in Table 1. Below are detailed descriptions of each case.
UBCU2-1
This previously healthy woman came from a family of European ancestry (Fig. 1) [19]. Her mother had ALS onset at age 51 and died at age 56 and her maternal uncle died in his 60s of ALS with dementia. At age 51, UBCU2-1 developed progressive, asymmetric upper limb weakness and occasional tripping. Initial examination found mild-to-moderate atrophy and weakness of upper and lower limb muscles, without fasciculations. Deep tendon reflexes were symmetrically brisk. Bulbar musculature was intact. Nerve conduction studies of median and ulnar nerves showed low amplitude but normal conduction velocities; whereas, sensory studies were normal. EMG showed denervation of several muscles in all four extremities and of paraspinal muscles. She was felt to be cognitively normal at that time but was not formally tested. A clinical diagnosis of definite ALS was made.
Over the subsequent 4 years, her extremity weakness progressed to the point of requiring full assistance for daily activities. She became increasingly dysarthric and had difficulty breathing, but was able to swallow. A change in personality was first noted 6 months before her death when she became disinhibited, mildly disoriented, inappropriately emotional and repetitive. She died at age 55 with a clinical diagnosis of ALS and early behavioral variant FTD (bvFTD). An autopsy limited to brain and spinal cord was performed. Neuropathological examination showed ALS-TDP and FTLD-TDP (subtype B) and tau-ir neurofibrillary tangles restricted to the entorhinal and transentorhinal cortex (Braak stage I).
UBCU2-2
The sister of the proband was assessed at age 55. Neither she nor her family reported any motor or cognitive symptoms. Clinical neurological assessment was normal; however, detailed neuropsychological assessment found deficits on measures of verbal and non-verbal memory. She was considered symptomatic (early memory abnormalities), but not demented.
UBCU2-14
The niece of the proband developed difficulty with word-finding at age 28. Around the same time, her family noted that she was becoming emotionally flat, withdrawn, apathetic and displaying little empathy. A few months later she developed dysarthria, difficulty chewing and swallowing and she became clumsy and prone to minor injury. Language comprehension began to decline, she was increasingly forgetful and had difficulty planning and organizing simple household tasks. She had suffered from dyslexia since childhood, as had several of her paternal relatives. When initially evaluated, 6–9 months following her disease onset, her Mini-Mental Status Examination (MMSE) score was 21/30, the Montreal Cognitive Assessment test (MoCA) showed prominent visuospatial dysfunction and the Frontal Assessment Battery (FAB) was 3/15. There was mild emotional incontinence. She had a slight spastic dysphonia and mild Gegenhalten in the right arm and leg. MRI demonstrated moderate symmetric frontotemporal atrophy.
When next evaluated, at age 29, she was found to have more severe cognitive impairment (MMSE score of 11/30 and MoCA score of 8/30). She was friendly but apathetic and emotionally blunt. Her speech was anomic, dysarthric and perseverative, but grammatically intact. She could follow simple commands and repeat sentences. Although her aphasia was difficult to classify, it was felt to have features of apraxia of speech and progressive non-fluent aphasia (PNFA). Physical examination was limited by her inability to fully cooperate. Strength of her facial muscles was normal, but tongue movement was impaired. Jaw jerk was brisk and she was hyperreflexic and mildly spastic in all extremities. Muscle bulk and strength were normal in all extremities and no fasciculations were noted. EMG showed active denervation, fibrillation potentials, irritability and some positive sharp waves in several muscles of both legs; however, due to patient noncompliance, the EMG study was discontinued before the upper extremities could be tested. The clinical diagnosis was that of primary progressive aphasia (PPA, difficult to classify) and “probable” ALS (due to the incomplete EMG study).
She progressed rapidly and became almost mute with a very limited degree of language comprehension within the next 6 months. Her bulbar dysfunction worsened and the right lower extremity became weak. She died at age 30 with a clinical diagnosis of FTD with both behavioral symptoms and progressive aphasia and probable ALS. Autopsy limited to brain and spinal cord demonstrated ALS-TDP and FTLD-TDP (subtype B).
NWU-1
This woman presented at age 65 with intermittent confusion and aphasia characterized by laconic speech, word finding difficulties and paraphasic errors in writing, but with intact language comprehension. No motor features were identified at that time and she was given a preliminary clinical diagnosis of primary progressive aphasia (PPA).
More detailed evaluation at age 67 found apraxia of speech, dysarthria, telegraphic phrases, anomia, problems with sentence comprehension and agrammatic writing. There were also impairments in executive function, motivation and insight. Motor examination demonstrated bulbar weakness, but normal limb strength and reflexes without fasciculations. MRI showed extensive cerebral white matter hyperintensities, attributed to chronic ischemia, and SPECT scan showed mild hypoperfusion of the left anterior temporal lobe.
Her disease progressed rapidly and by age 68 she had global aphasia, swallowing difficulties and fasciculations in the tongue and all limbs. EMG revealed findings of motor neuropathy and spontaneous motor activity, and swallowing studies were abnormal. She died later that year. Her family history was positive for late onset dementia, but not for ALS.
An autopsy was performed but was limited to the brain. Neuropathological examination showed FTLD-TDP (type B) and ALS-TDP pathology in the brainstem and high cervical spinal cord. There was very mild Alzheimer-type pathology with rare neuritic senile plaques and neurofibrillary tangles (Braak stage II).
TOR-1
This woman presented at age 73 with speech abnormalities characterized by frequent errors in grammar and syntax. Her speech progressively deteriorated and she also developed swallowing difficulties with frequent choking. Neuroimaging studies were unremarkable and she was diagnosed with PNFA and probable ALS. She developed depressive symptoms, but no other behavioral abnormalities. Her family history was negative for neurological disorders. Neuropathological examination showed ALS-TDP and FTLD-TDP (type B).
ALS701-01
This 75-year old woman presented with approximately 7 months of bulbar weakness and pseudobulbar affect and was diagnosed with clinically definite ALS. Her ALS Functional Rating Scale (ALSFRS) score was 25/48 and the ALS Cognitive Behavioral Screen (ALS-CBS) was compatible with probable frontotemporal cognitive impairment with expressive aphasia. She died at age 76. There was no family history of ALS, dementia or Parkinson disease. Post-mortem examination was not performed.
ALS458-01
This woman presented at age 63 with progressive bulbar weakness. By age 64 she met clinical criteria for definite ALS and required ventilatory support. Her ALSFRS score was 29/48. ALS-CBS was normal and she did not have any cognitive or behavioral symptoms. She was subsequently lost to follow up. Family history was positive for ALS and Parkinson disease.
ALS752-01
This woman presented at age 58 with 4 months of bulbar symptoms and was diagnosed with clinically definite ALS. Her ALSFRS score was 43/48 and ALS-CBS scores were normal. She died 21 months after disease onset. There was no family history of ALS, dementia or Parkinson disease. Autopsy limited to brain and spinal cord showed ALS-TDP and mild TDP-43 pathology in the extramotor cerebral cortex.
PITT-87
This woman presented at age 64 with sudden onset of bilateral leg weakness and back pain. EMG performed 4 months later showed widespread denervation in the legs and thoracic paraspinal muscles; however, weakness was only demonstrated in the distal legs. Over the following year, her weakness became more severe and spread to involve proximal legs, arms and face with a hyperactive jaw jerk and increased tone in the legs. Her respiratory function declined to a forced vital capacity of 33%. Cognitive testing performed at age 65 was normal with MMSE 30/30 and ALSCBS 19/20. She died at age 66. There was no history of neurological disorders in the family. Post-mortem examination was not performed.
Neuropathology
Gross pathology
The fresh brain weight ranged from 1132 to 1450 g (mean 1300 g) with two cases showing bifrontal lobar atrophy (UBCU2-1 and UBCU2-14) and one with left side predominant frontotemporal atrophy (TOR-1). The hippocampi were normal in size and only one case showed mild atrophy of the head of the caudate nucleus. Mild or moderate reduction in the pigmentation of the substantia nigra was noted in four cases.
General histology
The pyramidal motor system showed chronic degenerative changes in all cases (Table 2). The primary motor cortex tended to show mild neuronal loss and reactive changes, there was variable axonal and myelin loss in the corticospinal tracts (Fig. 2a) and moderate or severe loss of LMN in the brainstem and spinal cord. In all cases, small, brightly eosinophilic Bunina bodies were present in some of the remaining LMN (Fig. 2b). In addition, all cases were found to have some LMN containing sharply demarcated, round cytoplasmic inclusions that were often larger than the cell nucleus (Fig. 2c-e). These were pale pink or amphophilic with HE stain and approximately half had a compact central core, surrounded by a paler halo, similar in appearance to a Lewy body (Lewy body-like inclusion, LBLI). These round inclusions were distinct from the more irregularly shaped and more brightly eosinophilic hyaline inclusions that are frequently found in LMN in both ALS and normal aging (not shown). They were also easily identified on HE/LFB stained sections but did not stain with other histochemical stains such as Masson trichrome, Periodic Acid Schiff, Congo red or silver stains. Round inclusions were present in LMN of both the spinal cord ventral grey matter and the hypoglossal nucleus but were not seen in other neuronal populations. They averaged approximately two per tissue section, with a maximum frequency of 5 in one section (see quantitation below).
The four cases with clinical features of FTD also showed degeneration of the extramotor neocortex, with the prefrontal regions being most consistently and severely affected (Table 2, Fig. 2f). Apart from the pyramidal system motor nuclei, the only other subcortical regions that commonly showed degeneration were the caudate nucleus, periaqueductal grey matter and substantia nigra. The hippocampus was usually spared and no case showed selective loss of CA1 pyramidal neurons (hippocampal sclerosis).
TDP-43 immunoreactive pathology
TDP-ir pathology tended to be more severe and anatomically widespread than the degenerative changes (Table 2). There was moderate to severe involvement of the extramotor cerebral neocortex with the prefrontal cortex being most severely affected. In all cases, the pattern of pathology was most consistent with FTLD-TDP subtype B [18] with NCI in all cortical layers that were more often granular than compact (Fig. 3a). There were relatively few short thick dystrophic neurites (DN), but wispy threads and dot like structures were often concentrated in layer II. There were no neuronal intranuclear inclusions. Similar TDP-ir pathology was present in the primary motor cortex (Fig. 3b), but was milder in all cases with the exception of the patient with clinically pure ALS (ALS752-1) (Table 2). The hippocampus showed moderate numbers of TDP-ir NCI in the dentate granule cells (Fig. 3c) but few in the pyramidal layer and no TDP-ir wispy threads in the CA1/subiculum, characteristic of hippocampal sclerosis. Varying degrees of TDP-ir granular NCI and mild DN pathology was a consistent finding throughout the basal ganglia and substantia nigra and periaqueductal grey matter (Table 3, Fig. 3d); whereas, the thalamus and pons were only mildly and inconsistently involved and the cerebellum was spared.
Many of the remaining LMN in the spinal cord and hypoglossal nucleus contained TDP-ir NCI of varying morphology (Fig. 3e-i). Most common were small granules, diffusely distributed throughout the perikaryal cytoplasm (Figs. 3e, and 4). Filamentous NCI were also present (Fig. 3f), but were less common than large compact NCI that were often round and of similar size to the round/LBL inclusions seen on HE (Figs. 3g-i and 4). It was not uncommon to find a LMN that contained more than one inclusion type (Fig. 3i).
TDP-ir glial inclusions were not quantified separately but were a common feature in affected grey matter regions as well as the subcortical white matter. They were relatively uncommon in the corticospinal tracts and rare in other spinal cord funiculi.
Other pathological findings
Most cases did not show any other significant neurodegenerative pathology. Mild Alzheimer-type pathology was present in two cases with tau-ir neurofibrillary tangles limited to the entorhinal and transentorhinal cortex (Braak stage I or II, UBCU2-1 and NWU-1) and infrequent neuritic senile plaques (CERAD rare, NWU-1). No alpha-synuclein-ir Lewy bodies or neurites were present.
Comparison of pyramidal motor system pathology in ALS patients with and without TIA1 mutations
No differences were seen in the degree of chronic neurodegeneration in the primary motor cortex, corticospinal tracts, hypoglossal nucleus or ventral grey matter of the cervical or lumbar spinal cord when comparing ALS patients with TIA1 mutations, C9orf72 mutations and sALS without either mutation (LMN counts shown in Fig. 4a). There were also no differences in the burden of TDP-ir pathology in the primary motor cortex or the numbers of LMN containing Bunina bodies (data not shown). However, cases with TIA1 mutations had significantly more round inclusions and LBLI seen on HE stained sections of spinal cord than either the sALS or C9orf72+ group (Fig. 4b and c, p < 0.005 and p < 0.05, respectively). This difference was all the more striking given that the analysis included the TIA1 mutation case with only a single section of high cervical spinal cord available for examination (NWU-1). Although that case had only one round inclusion and no LBLI in the single cervical section (Fig. 4c, red circle), multiple round and LBLI were present in the hypoglossal nucleus. On average, one to two round inclusions were present in each section of spinal cord and medulla from the TIA1 mutation carriers; whereas, in the non-TIA1 cases, most sections did not have any round inclusions and cored LBLI were exceptionally rare.
With TDP-43 IHC, granular LMN NCI were the most frequent type in all three patient groups, representing 60-75% of the total. No differences were found among the groups in the frequency of granular or filamentous NCI (Fig. 4d and e); however, the TIA1 mutation cases had significantly more compact NCI (p < 0.05) than C9orf72+ or sALS cases (Fig. 4f).
Immunostaining for TIA1, other SG components and other RNA-binding proteins (RBP)
IHC using a number of anti-TIA1 primary antibodies that recognize different TIA1 epitopes (Table 3) failed to demonstrate any abnormality. Most of the antibodies showed moderately intense diffuse staining of neuronal cytoplasm and some also stained the nucleus, although none showed preferential nuclear staining (Fig. 5a). The staining patterns were similar in ALS cases with and without TIA1 mutations and in sections from normal controls. Specifically, no TIA1-ir pathological inclusions were demonstrated in cases with TIA1 mutations. For double label IF an antibody that recognizes phosphorylated pathological TDP-43 (pTDP-43) was used (to avoid the normal nuclear positivity) in combination with each of the three TIA1 antibodies that gave the best results with IHC. The same types of NCI were labelled for pTDP-43 as had been seen on light microscopy with TDP-43 IHC (Fig. 5b-d). The TIA1 antibodies again showed diffuse cytoplasmic +/− nuclear reactivity; however, there was no specific co-localization of TIA1 with compact, filamentous or granular pTDP-43-ir NCI (Fig. 5b-d). IHC using antibodies against another SG marker (PABP) and against a number of other RBP (FUS, hnRNPA1, hnRNPA3 and hnRNPA2B1) also failed to show any distinctive staining pattern in the TIA1 mutation cases (data not shown).